Energy aware sensor management for wearable medical systems optimization

ABSTRACT

In some aspects, the invention provides compositions and methods for inhibiting viral infection. In some aspects, the invention provides compositions and methods useful for identifying antiviral compounds.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ApplicationNo. 61/356,426, filed Jun. 18, 2010. The entire contents of thisapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Viruses are major causes of disease and death throughout the world.Although vaccines and public health measures have greatly reduced theincidence of certain viral infections, such approaches have been lesssuccessful in tackling many viruses of significant medical and/orveterinary importance. Even if a generally protective vaccine exists, itis challenging to achieve vaccination of all individuals. Furthermore,obstacles to effective immunization can arise due to factors such asimmune senescence and treatment with immunosuppressive medications.Pharmacological therapies have been developed against some viruses, withhuman immunodeficiency virus (HIV) being a notable example. However,there are still relatively few viral diseases for which effective drugsare available. There is a need for new antiviral compounds and for newapproaches to identifying such compounds.

SUMMARY OF THE INVENTION

The invention relates at least in part to identification of a target forantiviral drug discovery. In one aspect, the invention provides a methodof inhibiting viral infection of a cell comprising contacting the cellwith a PLA2G16 inhibitor. In some embodiments, the virus is aPicornavirus. In some embodiments, the cell is a vertebrate cell. Insome embodiments the vertebrate cell is a mammalian cell, e.g., a humancell. In some embodiments, the PLA2G16 inhibitor inhibits expression ofPLA2G16. In some embodiments, the PLA2G16 inhibitor inhibits enzymaticactivity of PLA2G16.

In another aspect, the invention provides a method of treating a viralinfection in a subject, the method comprising administering a PLA2G16inhibitor to a subject in need of treatment for a viral infection. Insome embodiments, the viral infection is a Picornavirus infection. Insome embodiments, the subject is a vertebrate. In some embodiments, thesubject is a mammal, e.g., a human. In some embodiments, the PLA2G16inhibitor inhibits expression of PLA2G16. In some embodiments, thePLA2G16 inhibitor inhibits enzymatic activity of PLA2G16.

In another aspect, the invention provides a method of identifying acandidate antiviral compound comprising steps of: (a) providing acomposition comprising a PLA2G16 polypeptide and a test compound; (b)determining whether the test compound inhibits the PLA2G16 polypeptide,wherein if the compound inhibits the PLA2G16 polypeptide, the compoundis identified as a candidate antiviral compound. In some embodiments,step (b) comprises determining whether the test compound inhibitsexpression of the PLA2G16 polypeptide. In some embodiments, step (b)comprises determining whether the test compound inhibits an enzymaticactivity of the PLA2G16 polypeptide. In some embodiments, the enzymaticactivity is phospholipase A2 activity. In some embodiments, thecomposition of step (a) is a cell-free composition comprising purifiedPLA2G16; and step (b) comprises determining whether the test compoundinhibits enzymatic activity of PLA2G16. In some embodiments, thecomposition of step (a) comprises a cell that expresses a PLA2G16polypeptide, and wherein step (b) comprises determining whether the testcompound inhibits expression or enzymatic activity of PLA2G16. In someembodiments, if the compounds inhibits the PLA2G16 polypeptide, thecompound is identified as a candidate antiviral compound useful forinhibiting viral infection by a Picornavirus. In some embodiments, themethod further comprises assessing the ability of the compound toinhibit viral infection of a cell or subject. In some embodiments, themethod further comprises the step of contacting a cell with the compoundand a virus, wherein the cell would be susceptible to the virus in theabsence of the compound. In some embodiments, the method furthercomprises the step of administering the compound to a subject, whereinthe subject would be susceptible to infection by the virus in theabsence of the compound. In some embodiments, the method furthercomprises the step of contacting a cell that is infected by the viruswith the compound. In some embodiments, the method further comprises thestep of administering the compound to a subject, wherein the subject isinfected by a virus.

In another aspect, the invention provides a method of validating acandidate antiviral compound comprising steps of: (a) providing acandidate antiviral compound identified according to a method thatcomprises identifying or selecting a compound that inhibits PLA2g16; and(b) determining whether the compound inhibits infection of a cell ororganism by a virus, wherein if the compound inhibits infection of acell or organism by the virus, the compound is validated as an antiviralcompound. In some embodiments, the virus is a Picornavirus.

In another aspect, the invention provides a composition comprising: (a)a PLA2G16 inhibitor; (b) a virus; and (c) a population of cells. In someembodiments, the virus is present at a multiplicity of infection (MOI)of at least 0.01. In some embodiments, the virus is a Picornavirus. Insome embodiments, the cells are in culture. In some embodiments, thecells are vertebrate cells. In some embodiments, the cells are mammaliancells, e.g., human cells. In some embodiments, the population of cellscomprises at least 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, or more cells. In someembodiments, the cells are human cells. In some embodiments, at leastsome of the cells are infected by the virus. In some embodiments, thePLA2G16 inhibitor binds to PLA2G16. In some embodiments, the PLA2G16inhibitor inhibits expression of PLA2G16. In some embodiments, thePLA2G16 inhibitor inhibits an enzymatic activity of PLA2G16. In someembodiments, the PLA2G16 inhibitor is a small molecule. In someembodiments, the PLA2G16 inhibitor is present in an amount sufficient todetectably inhibit infection of the cells by the virus.

In another aspect, the invention provides a composition comprising aPLA2G16 inhibitor, wherein the composition is useful for treating aviral infection in a subject. In some embodiments, the PLA2G16 inhibitorbinds to PLA2G16. In some embodiments, the PLA2G16 inhibitor inhibitsexpression of PLA2G16. In some embodiments, the PLA2G16 inhibitorinhibits an enzymatic activity of PLA2G16. In some embodiments, thePLA2G16 inhibitor is a small molecule. In some embodiments, the viralinfection is a Picornavirus infection. In some embodiments, the subjectis a vertebrate. In some embodiments, the subject is a mammal, e.g., ahuman.

In another aspect, the invention provides a mammalian cell that has amutation in a gene that encodes PLA2G16. In some embodiments the cell isa near-haploid cell. In some embodiments, the cell expresses a mutantform of PLA2G16. In some embodiments, the cell expresses a mutant formof PLA2G16, wherein the mutant form has reduced catalytic activity ascompared with the non-mutant form.

In another aspect, the invention provides a method of identifying anon-human multicellular organism, e.g., a vertebrate animal, that hasincreased resistance to viral infection, the method comprisingidentifying a multicellular organism that has reduced or absentfunctional PLA2G16. In some embodiments the invention provides a methodof identifying a non-human multicellular organism with increasedresistance to infection by a virus, the method comprising determiningwhether the organism has reduced PLA2G16 expression or activity, whereinif the organism has reduced PLA2G16 expression or activity, the organismhas increased resistance to infection by a virus. In some embodiments,the method further comprises providing or using an organism with reducedor absent PLA2G16 in agriculture and/or animal husbandry. In someembodiments, a virus-resistant animal is of a non-domesticated species.Optionally the species is endangered. In some embodiments, the organismis a commercially important vertebrate animal. In some embodiments ofthe inventive methods, the organism is not genetically modified.

In another aspect, the invention provides a farm animal having reducedor absent functional PLA2G16, wherein the animal has increasedresistance to infection by a virus. In some embodiments the animal isnot genetically modified. In other embodiments the animal is geneticallymodified.

In certain embodiments of any of the aspects of the invention, thePicornavirus is an enterovirus (member of the Enterovirus genus). Incertain embodiments the enterovirus is a human enterovirus, e.g., avirus classified within the Human enterovirus A, Human enterovirus B,Human enterovirus C, Human enterovirus D, Human rhinovirus A, Humanrhinovirus B, or Human rhinovirus C species. In some embodiments, thehuman enterovirus is poliovirus 1, 2, or 3 or any of human enteroviruses68-107, e.g., EV-71. In certain embodiments of any of the aspects of theinvention, the Picornavirus is a hepatovirus, e.g., human hepatitis Avirus. In certain embodiments of any of the aspects of the invention,the Picornavirus is a coxsackievirus. In certain embodiments thecoxsackievirus is a human coxsackievirus, e.g., any coxsackievirusesA1-A22, A24, or B1-B5. In certain embodiments of any of the aspects ofthe invention, the Picornavirus is a rhinovirus (member of Humanrhinovirus A, Human rhinovirus B, or Human rhinovirus C species), e.g.,any of human rhinoviruses 1-100. In certain embodiments of any of theaspects of the invention, the Picornavirus is an echovirus. In certainembodiments of any of the various aspects of the invention, the virus isa foot-and-mouth disease virus, e.g., one of the seven foot-and-mouthdisease virus serotypes: O, A, C, SAT-1, SAT-2, SAT-3, and Asia-1.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding viruses is foundin, e.g., Knipe, D M and Howley, P M (eds.) Fields Virology, Volumes Iand II. 5^(th) ed. Lippincott Williams and Wilkins, 2007; Büchen-Osmond,C. (Ed), (2006) Index to ICTVdB virus descriptions. In: ICTVdB—TheUniversal Virus Database, version 4. ICTVdB Management, Mailman Schoolof Public Health, Columbia University, New York, N.Y., USA; and“ICTVdB—The Universal Virus Database”, version 4, April 2006.http://www.ictvdb.org/Ictv/ICTVindex.htm) and ICTVdb Virus Descriptions(http://www.ictvdb.org/ICTVdB/index.htm). (It is noted that the onlinedatabase is currently being rewritten.) The most recent report of theInternational Committee on the Taxonomy of Viruses (ICTV) of theInternational Union of Microbiological Societies: “Virus Taxonomy:VIIIth Report of the International Committee on Taxonomy of Viruses”,2005, C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A.Ball (Eds), Elsevier Academic Press, is considered the standard anddefinitive reference for virus taxonomy (classification andnomenclature), as supplemented by taxonomic proposals subsequentlyapproved by the ICTV (available as updates on the ICTV website ashttp://talk.ictvonline.org/media/22/default.aspx/.http://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the_(—)8th_report/default.aspx)(See also Carstens, E B and Ball, L. Ratification vote on taxonomicproposals to the International Committee on Taxonomy of Viruses.Archives of Virology, Volume 154, Number 7, 2008, and Carstens, E.Ratification vote on taxonomic proposals to the International Committeeon Taxonomy of Viruses (2009) Archives of Virology, Volume 155, Number1, 2009). The Virus Taxonomy: 2009 Release v4 (Mar. 20, 2010) (availableon the ICTV website at http://ictvonline.org/virusTaxonomy.asp)represents the most recent taxonomy.

Non-limiting information regarding therapeutic agents and human diseasesis found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology; McGraw-Hill/Appleton & Lange; 10^(th) ed. (2006)or 11th edition (July 2009). All patents, patent applications, and otherpublications (e.g., scientific articles, books, websites, and databases)mentioned herein are incorporated by reference in their entirety. Incase of a conflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A. Schematic outline of gene-trap vector integration in anendogenous gene. B. Schematic overview of haploid genetic screen forgenes critical for poliovirus replication.

FIG. 2. Haploid genetic screen identifies PLA2G16 as critical forpoliovirus infection. Mutagenized haploid cells were contacted withpoliovirus and resistant colonies were allowed to grow out. Gene trapinsertion sites were determined using inverse PCR and massively parallelsequencing. The plot shows the positions on the human chromosome towhich individual gene trap mutations were mapped on the x-axis and theinverse of the distance of a particular mutation to its neighbors on they-axis. Mutations are highly enriched in chromosome 19 in the knownpoliovirus receptor (PVR) and on chromosome 11 in the phospholipasePLA2G16 that contained 42 independent gene trap insertions.

FIG. 3. Western blot analysis for expression of PLA2G16 in wild typehaploid cells (WT; lane 1), cells containing a gene trap insertion inPLA2G16 gene (PLA2G16^(GT:); lane 2) cells containing a gene trap inPLA2G16 and expressing FLAG-tagged PLA2G16 (lane 3); cells containing agene trap in PLA2G16 expressing FLAG-tagged mutant PLA2G16 (lane 4);cells containing a gene trap in PLA2G16 and expressing untagged PLA2G16(lane 5); cells containing a gene trap in PLA2G16 and expressinguntagged mutant PLA2G16 (lane 6).

FIG. 4. Haploid cells containing a PLA2G16 gene trap insertion areresistant to poliovirus infection. Complementation of PLA2G16 byretroviral overexpression restores sensitivity of these cells topoliovirus. This requires the catalytic activity of PLA2G16 becausecomplementation with a catalytic site mutant (C113A) does not restoresensitivity.

FIG. 5. Cells containing a PLA2G16 gene trap insertion are resistant tocoxsackievirus B1. Cells were plated in 24-well wells and monolayerswere virus was added at the indicated MOIs. Four days after infectionviable, adherent cells were stained using crystal violet. Cells mutantfor PLA2G16 were unaffected by high concentrations of coxsackievirus B1.Complementation of PLA2G16 by retroviral overexpression restoressensitivity of these cells to coxsackievirus B1. This requires thecatalytic activity of PLA2G16 because complementation with a catalyticsite mutant (C113A) does not restore sensitivity.

FIG. 6. (A) Sensitivity of wild type and gene trap mutant cells topoliovirus. (B) Sensitivity of wild type gene trap mutant cells tocoxsackievirus B1. Poliovirus was added to cells at the indicated MOIs(X-axis) and viability was measured three days later using an MTT assay.HAP1: Wild type HAP1 cells (without gene trap)

-   PLA2G16: HAP1 cells containing gene trap insertion into PLA2G16 gene    (PLA2G16^(GT))-   PLA2G16+PM2G16WT: HAP1 PLA2G16^(GT) cells infected with retrovirus    encoding wild type PLA2G16-   PLA2G16+PM2G16MUT: HAP1 PLA2G16^(GT) cells infected with retrovirus    encoding catalytically inactive mutant PLA2G16 (with C113A mutation)-   PVR: HAP1 cells with gene trap insertion into poliovirus receptor.

FIG. 7. Knock down of PLA2G16 in Hela cells results in increasedresistance to the human rhinoviruses HRV-2 and HRV-14.

FIG. 8. Exemplary PLA2G16 sequences. Predicted transmembrane domain isshown in bold in the human sequence.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION I.Definitions

The term “antibody” encompasses immunoglobulins and derivatives thereofcontaining an immunoglobulin domain capable of binding to an antigen. Anantibody can originate from a mammalian or avian species, e.g., human,rodent (e.g., mouse, rabbit), goat, chicken, etc., or can be generatedex vivo using a technique such as phage display. Antibodies includemembers of the various immunoglobulin classes, e.g., IgG, IgM, IgA, IgD,IgE, or subclasses thereof such as IgG1, IgG2, etc. In variousembodiments of the invention “antibody” refers to an antibody fragmentor molecule such as an Fab′, F(ab′)2, scFv (single-chain variable) thatretains an antigen binding site and encompasses recombinant moleculescomprising one or more variable domains (VH or VL). An antibody can bemonovalent, bivalent or multivalent in various embodiments. The antibodymay be a chimeric or “humanized” antibody. An antibody may be polyclonalor monoclonal, though monoclonal antibodies may be preferred. In someaspects, an antibody is an intrabody, which may be expressedintracellularly. In some embodiments a compound comprises a single-chainantibody and a protein transduction domain (e.g., as a fusionpolypeptide).

An “effective amount” or “effective dose” of a compound or other agent(or composition containing such compound or agent) refers to the amountsufficient to achieve a desired biological and/or pharmacologicaleffect, e.g., when delivered to a cell or organism according to aselected administration form, route, and/or schedule. As will beappreciated by those of ordinary skill in this art, the absolute amountof a particular compound, agent, or composition that is effective mayvary depending on such factors as the desired biological orpharmacological endpoint, the agent to be delivered, the target tissue,etc. Those of ordinary skill in the art will further understand that an“effective amount” may be contacted with cells or administered in asingle dose, or the desired effect may be achieved by use of multipledoses. An effective amount of an antiviral compound may be an amountsufficient to achieve one or more of the following: (i) reduce virusreplication (e.g., reduce production of progeny virus) in cell cultureand/or in vivo; (ii) reduce the severity of or prevent one or moresymptoms or signs of a viral infection; (iii) significantly reduce therisk of recurrence of a viral infection (e.g., reduce the risk ofrelapse); (iv) significantly reduce the risk of a clinically significantinfection in a subject who has been exposed to an infectious agent, etc.

“Identity” or “percent identity” is a measure of the extent to which thesequence of two or more nucleic acids or polypeptides is the same. Thepercent identity between a sequence of interest A and a second sequenceB may be computed by aligning the sequences, allowing the introductionof gaps to maximize identity, determining the number of residues(nucleotides or amino acids) that are opposite an identical residue,dividing by the minimum of TG_(A) and TG_(B) (here TG_(A) and TG_(B) arethe sum of the number of residues and internal gap positions insequences A and B in the alignment), and multiplying by 100. Whencomputing the number of identical residues needed to achieve aparticular percent identity, fractions are to be rounded to the nearestwhole number. Sequences can be aligned with the use of a variety ofcomputer programs known in the art. For example, computer programs suchas BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., generate alignments. Thealgorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad.Sci. USA 87:22264-2268, 1990) modified as in Karlin and Altschul, Proc.Natl. Acad Sci. USA 90:5873-5877, 1993 is incorporated into the NBLASTand XBLAST programs of Altschul et al. (Altschul, et al., J. Mol. Biol.215:403-410, 1990). In some embodiments, to obtain gapped alignments forcomparison purposes, Gapped BLAST is utilized as described in Altschulet al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402, 1997). Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs may be used. See the Web site having URLwww.ncbi.nlm.nih.gov. Other suitable programs include CLUSTALW (ThompsonJ D, Higgins D G, Gibson T J, Nuc Ac Res, 22:4673-4680, 1994) and GAP(GCG Version 9.1; which implements the Needleman & Wunsch, 1970algorithm (Needleman S B, Wunsch C D, J Mol Biol, 48:443-453, 1970.)

“Infection” refers to the (often detrimental) colonization of a cell(sometimes referred to as a “host cell” or “host”) or multicellularorganism (sometimes referred to as a “host”), by a microorganism such asa virus. The process of infection encompasses entry of the virus intoone or more cell(s) (invasion) and, if the infection proceeds,subsequent steps in the viral life cycle, typically resulting inmultiplication of the virus and, frequently in the case of viruses ofmedical or veterinary importance, detrimental effects of the virus onthe host. A viral infection can be any situation in which the presenceof one or more virus population(s) is damaging to a host cell ororganism. The term “infection” encompasses excessive replication ofviruses that are normally present in or on the body of a vertebrate,e.g., mammal, or other organism, or the presence and, optionally,replication, of viruses that are not normally present in or on the bodyof a vertebrate, e.g., a mammal, or other organism.

“Inhibit” may be used interchangeably with terms such as “suppress”,“decrease”, and the like, as appropriate in the context. It will beunderstood that the extent of inhibition can vary. For example,inhibition can refer to a reduction by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99%.

“Isolated” refers to a substance that is separated from at least someother substances with which it is normally found in nature, usually by aprocess involving the hand of man, or is artificially produced, e.g.,chemically synthesized, or present in an artificial environment.

“Nucleic acid” is used interchangeably with “polynucleotide” andencompasses naturally occurring polymers of nucleosides, such as DNA andRNA, usually linked by phosphodiester bonds, and non-naturally occurringpolymers of nucleosides or nucleoside analogs. In some embodiments anucleic acid comprises standard nucleotides (abbreviated A, G, C, T, U).In other embodiments a nucleic acid comprises one or more non-standardnucleotides. In some embodiments, one or more nucleotides arenon-naturally occurring nucleotides or nucleotide analogs. A nucleicacid can comprise chemically or biologically modified bases (forexample, methylated bases), modified sugars (2′-fluororibose, arabinose,or hexose), modified phosphate groups (for example, phosphorothioates or5′-N-phosphoramidite linkages), locked nucleic acids, or morpholinos. Insome embodiments, a nucleic acid comprises nucleosides that are linkedby phosphodiester bonds. In some embodiments, at least some nucleosidesare linked by a non-phosphodiester bond. A nucleic acid can besingle-stranded, double-stranded, or partially double-stranded. An atleast partially double-stranded nucleic acid can have one or moreoverhangs, e.g., 5′ and/or 3′ overhang(s). Nucleic acid modifications(e.g., nucleoside and/or backbone modifications), non-standardnucleotides, delivery vehicles and approaches, etc., known in the art asbeing useful in the context of RNA interference (RNAi), aptamer, orantisense-based molecules for research or therapeutic purposes arecontemplated for use in various embodiments of the instant invention.See, e.g., Crooke, S T (ed.) Antisense drug technology: principles,strategies, and applications, Boca Raton: CRC Press, 2008; Kurreck, J.(ed.) Therapeutic oligonucleotides, RSC biomolecular sciences.Cambridge: Royal Society of Chemistry, 2008. A nucleic acid may comprisea detectable label, e.g., a fluorescent dye, radioactive atom, etc.“Oligonucleotide” refers to a relatively short nucleic acid, e.g.,typically between about 4 and about 60 nucleotides long.

A “polypeptide” refers to a polymer of amino acids linked by peptidebonds. A protein is a molecule comprising one or more polypeptides. Apeptide is a relatively short polypeptide, typically between about 2 and60 amino acids in length. The terms “protein”, “polypeptide”, and“peptide” may be used interchangeably. Polypeptides of interest hereinoften contain standard amino acids (the 20 L-amino acids that are mostcommonly found in nature in proteins). However, other amino acids and/oramino acid analogs known in the art can be used in certain embodimentsof the invention. One or more of the amino acids in a polypeptide (e.g.,at the N- or C-terminus or in a side chain) may be modified, forexample, by addition, e.g., covalent linkage, of a moiety such as analkyl group, carbohydrate group, a phosphate group, a halogen, a linkerfor conjugation, etc. A polypeptide sequence presented herein ispresented in an N-terminal to C-terminal direction unless otherwiseindicated. “Polypeptide domain” refers to a segment of amino acidswithin a longer polypeptide. A polypeptide domain may exhibit one ormore discrete binding or functional properties, e.g., a catalyticactivity. Often a domain is recognizable by its conservation amongpolypeptides found in multiple different species.

As used herein, the term “purified” refers to agents or entities (e.g.,compounds) that have been separated from most of the components withwhich they are associated in nature or when originally generated. Ingeneral, such purification involves action of the hand of man. Purifiedagents or entities may be partially purified, substantially purified, orpure. Such agents or entities may be, for example, at least 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% pure.In some embodiments, a nucleic acid or polypeptide is purified such thatit constitutes at least 75%, 80%, 855%, 90%, 95%, 96%, 97%, 98%, 99%, ormore, of the total nucleic acid or polypeptide material, respectively,present in a preparation. Purity can be based on, e.g., dry weight, sizeof peaks on a chromatography tracing, molecular abundance, intensity ofbands on a gel, or intensity of any signal that correlates withmolecular abundance, or any art-accepted quantification method. In someembodiments, water, buffers, ions, and/or small molecules (e.g.,precursors such as nucleotides or amino acids), can optionally bepresent in a purified preparation. A purified molecule may be preparedby separating it from other substances (e.g., other cellular materials),or by producing it in such a manner to achieve a desired degree ofpurity. In some embodiments, a purified molecule or composition refersto a molecule or composition that is prepared using any art-acceptedmethod of purification. In some embodiments “partially purified” meansthat a molecule produced by a cell is no longer present within the cell,e.g., the cell has been lysed and, optionally, at least some of thecellular material (e.g., cell wall, cell membrane(s), cell organelle(s))has been removed.

“RNA interference” (RNAi) encompasses processes in which an endogenousmolecular complex known as an RNA-induced silencing complex (RISC)silences gene expression in a sequence-specific manner. The RISCcontains a short RNA strand that directs or “guides” sequence-specificdegradation or translational repression of mRNA to which it hascomplementarity. The complementarity between the short RNA and mRNA neednot be perfect (100%). For example, the degree of complementarity and/orthe characteristics of the structure formed by hybridization of the mRNAand the short RNA strand can be such that the strand can (i) guidecleavage of the mRNA in the RNA-induced silencing complex (RISC) and/or(ii) cause translational repression of the mRNA by RISC. It will beappreciated that one or more mismatches between the guide strand and thetarget mRNA can be tolerated, especially outside the seed region (thenucleotides in positions 2-7 or 2-8) of the guide strand. A short RNAthat guides silencing often initially becomes associated with RISCcomponents (in a complex sometimes termed the RISC loading complex) aspart of a short double-stranded RNA (dsRNA). RNAi is often used toknockdown a target gene. “Knockdown” typically refers to a reduction inexpression, which may occur, e.g., at the level of transcription, mRNAstability, translation, or protein stability. Reduction can be complete(e.g., the amount of gene product is reduced to background levels) orless than complete. For example, mRNA and/or protein level can bereduced by 50%, 60%, 70%, 75%, 80%, 85%, 90%, or more.

RNAi may be employed to inhibit expression in eukaryotic cells, e.g.,vertebrate cells, in a variety of ways as known in the art. In someembodiments, a short double-stranded nucleic acid is introduced intocells. In some embodiments, a nucleic acid that is processedintracellularly (e.g., by one or more RNase III family enzymes Dicer) toyield short dsRNA is introduced into or expressed in cells. As usedherein, the term “RNAi agent” encompasses nucleic acids that can be usedto achieve RNAi in eukaryotic cells. Exemplary RNAi agents are shortinterfering RNA (siRNA) and short hairpin RNA (shRNA). As known in theart, siRNAs typically comprise two separate nucleic acid strands thatare hybridized to each other to form a duplex. They can be synthesizedin vitro, e.g., using standard nucleic acid synthesis techniques or bycleavage of a longer dsRNA, e.g., by an RNase III or RNase III-likeenzyme such as Dicer. In certain embodiments an siRNA or shRNA comprisesa duplex portion about 15-29 nucleotides (nt) long, e.g., between 17-25nt long, e.g., between 19-23 nt long, wherein either or both strandsoptionally has a 3′ overhang of 1-5 nucleotides long (e.g., 2nucleotides), which may be composed of deoxyribonucleotides. In someembodiments, the strands are perfectly complementary within the duplexportion, while in other embodiments, the duplex portion could containone or more mismatched nucleotide pairs or bulges. In some embodiments,each strand of an siRNA is between 15-29 nucleotides in length, e.g.,between 19-25 nt long, e.g., 21-23 nt long. shRNA comprise a singlenucleic acid strand that contains two complementary portions separatedby a predominantly non-self-complementary region. The complementaryportions hybridize to form a duplex structure and thenon-self-complementary region forms a loop connecting the 3′ end of onestrand of the duplex and the 5′ end of the other strand. shRNAs canundergo intracellular processing to generate siRNAs.

RNAi agents also include microRNA (miRNA) and miRNA precursors. Theterms “miRNA” and “miRNA precursor” are often used in the art to referto endogenously encoded RNAs. As used herein, “miRNA” and “miRNAprecursor” encompasses artificially designed nucleic acids that functionin an analogous manner to endogenous miRNAs.

In certain embodiments an RNAi agent is a vector that comprises atemplate for transcription of an siRNA (e.g., as two separate strandsthat can hybridize to each other), shRNA, or microRNA precursor. Suchvectors can be used to introduce the template into vertebrate cells,e.g., mammalian cells, and result in transient or stable expression ofthe siRNA, shRNA, or miRNA precursor.

A “small molecule” as used herein, is an organic molecule that is lessthan about 2 kilodaltons (KDa) in mass. In some embodiments, the smallmolecule is less than about 1.5 KDa, or less than about 1 KDa. In someembodiments, the small molecule is less than about 800 daltons (Da), 600Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small moleculehas a mass of at least 50 Da. In some embodiments, a small molecule isnon-polymeric. In some embodiments, a small molecule is not an aminoacid. In some embodiments, a small molecule is not a nucleotide. In someembodiments, a small molecule is not a saccharide. In some embodiments,a small molecule contains multiple carbon-carbon bonds and can compriseone or more heteroatoms and/or one or more functional groups importantfor structural interaction with proteins (e.g., hydrogen bonding), e.g.,an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodimentsat least two functional groups. Small molecules often comprise one ormore cyclic carbon or heterocyclic structures and/or aromatic orpolyaromatic structures, optionally substituted with one or more of theabove functional groups.

A “subject” can be any multicellular organism, e.g., a multicellularorganism that is susceptible to infection by a virus or is or may beinfected by a virus. Often at least some of the cells of the subjectexpress detectable amounts of PLA2G16. In some embodiments a subject isan animal, e.g., a vertebrate, e.g., a mammal or avian. Exemplarymammals include, e.g., humans, non-human primates, rodents (e.g., mouse,rat, rabbit), ungulates (e.g., ovine, bovine, equine, caprine species),canines, and felines. In some embodiments, the animal is a mammal ofeconomic importance, such as a cow, horse, pig, goat, or sheep. Often, asubject is an individual to whom a compound is to be delivered, e.g.,for experimental, diagnostic, and/or therapeutic purposes or from whom asample is obtained or on whom a diagnostic procedure is performed (e.g.,a sample or procedure that will be used to determine whether the subjecthas a viral infection or is at risk of a viral infection).

“Treat”, “treating” and similar terms refer to providing medical and/orsurgical management of a subject. Treatment can include, but is notlimited to, administering a compound or composition (e.g., apharmaceutical composition) to a subject. Treatment is typicallyundertaken in an effort to alter the course of a disease, disorder, orundesirable condition in a manner beneficial to the subject. The effectof treatment can generally include reversing, alleviating, reducingseverity of delaying the onset of, curing, inhibiting the progressionof, and/or reducing the likelihood of occurrence or reoccurence of thedisease, disorder, or condition to which such term applies, or one ormore symptoms or manifestations of such disease, disorder or condition.A composition of this invention can be administered to a subject who hasdeveloped an infection or is at increased risk of developing aninfection relative to a member of the general population. A compositionof this invention can be administered prophylactically, i.e., beforedevelopment of any symptom or manifestation of a condition. Typically inthis case the subject will be at risk of developing the condition. Forexample, an inventive composition can be administered prior to exposureof the subject to an infectious agent or prior to the occurrence of apathogenic event. “Preventing” can refer to administering a compound orcomposition (e.g., a pharmaceutical composition) to a subject who hasnot developed a disease or condition, so as to reduce the likelihoodthat the disease or condition will occur or so as to reduce the severityof the disease or condition should it occur. The subject may beidentified as at risk of developing the disease or condition (e.g., atincreased risk relative to many most other members of the population oras having a risk factor that increases likelihood of developing thedisease).

II. Overview

The present invention relates in part to the identification ofphospholipase A2, group XVI (PLA2G16) as a new molecular target of usefor identification and/or characterization of antiviral compounds.PLA2G16 is a phospholipase that is widely or ubiquitously expressed inmammalian tissues. It has now been discovered that PLA2G16 polypeptideis a host cell factor that plays an important role in infection ofvertebrate cells, e.g., mammalian cells, by viruses of the Picornavirusfamily. The invention encompasses the recognition that inhibitingPLA2G16 inhibits viral infection. As described in more detail in theExamples, using a gene trap mutagenesis strategy in a near-haploidmammalian cell line (the HAP1 cell line), it was shown that insertionsinto the PLA2G16 gene (located on chromosome 11 in human cells) renderedthe cells resistant to infection by poliovirus and Coxsackie virus B1.Restoring wild type PLA2G16 function by expressing wild type PLA2G16 inthe cells restored susceptibility to infection, while expressing acatalytically inactive mutant version of PLA2G16 did not. Furthermore,knockdown of endogenous PLA2G16 expression in a rhinovirus-sensitivecell line (HeLa cells) using short interfering RNA (siRNA) renderedthese cells resistant to rhinovirus infection. The discoveries describedherein indicate that PLA2G16 is required for infection of vertebratecells by a wide range of viruses.

The invention provides compositions and methods for inhibiting viralinfection. The invention further provides compositions and methodsuseful for identifying candidate compounds for inhibiting viralinfection. In some aspects, the compositions and methods relate to theuse of the PLA2G16 gene and/or PLA2G16 polypeptide as targets foridentification of antiviral compounds (i.e., compounds that inhibitviral infection). Certain of the inventive methods comprise identifyingor providing a compound that inhibits PLA2G16. In accordance withcertain embodiments of the invention, a compound that inhibits PLA2G16is a candidate antiviral compound. Certain of the inventive methodscomprise (i) identifying or providing a compound that inhibits PLA2G16;and (ii) determining whether the compound inhibits viral infection of acell or multicellular organism, wherein if the compound inhibits viralinfection of a cell or multicellular organism, the compound is anantiviral compound. In some embodiments, a compound that inhibitsPLA2G16 inhibits PLA2G16 expression. In some embodiments, a compoundthat inhibits PLA2G16 inhibits a PLA2G16 molecular function, e.g., thecompound inhibits PLA2G16 catalytic activity.

Inhibiting viral infection can comprise interventions that inhibit oneor more steps of the viral life cycle so that, for example, there isreduced entry of virus into cells, reduced production of viral geneproduct(s) (viral RNAs and/or proteins), reduced production of progenyvirus, reduced release of progeny virus, and/or reduced spread of viruswithin a population of cells (e.g., in cell culture or in amulticellular orgnanism) as compared with an appropriate referencelevel, e.g., the level that would exist in the absence of theintervention. Inhibition of viral infection can be assessed based on anyof a variety of suitable indicators. In some embodiments, inhibition ofan indicator of viral infection is complete or substantially complete,e.g., an indicator of viral infection such as production of a viral geneproduct, production of progeny virus, infection of additional cells, isreduced to background or undetectable level, e.g., a level that would beexpected in the absence of the virus. In some embodiments, inhibition isnot complete. In some embodiments, inhibition of viral infection canrefer to a reduction by about a factor of at least 10, at least 10², atleast 10³, at least 10⁴, or more, e.g., in production of progeny virusor of a viral gene product.

In some aspects, the invention provides methods of inhibiting viralinfection of a cell. In some aspects, the methods comprise inhibitingPLA2G16 in a cell, thereby inhibiting viral infection of the cell. Insome embodiments, the methods comprise contacting a cell with a compoundthat inhibits PLA2G16, so that viral infection of the cell is inhibited.In some embodiments, the cell is an animal cell, e.g., a vertebratecell. In some embodiments, the vertebrate cell is a mammalian cell. Insome aspects, the invention provides methods of inhibiting viralinfection of a subject. In some embodiments, the subject is avertebrate. In some aspects, the methods comprise inhibiting PLA2G16 inat least some cells of the organism, e.g., at least some cells that areinfected by a virus or are susceptible to infection by a virus. In someembodiments, the methods comprise administering a compound that inhibitsPLA2G16 to the subject. In some embodiments, the subject is an animal,e.g., a vertebrate. In some embodiments, the vertebrate is a mammal.

In some aspects, the invention provides methods of decreasing thesusceptibility (or increasing the resistance) of a cell or subject to avirus, the methods comprising inhibiting PLA2G16 in a cell or in atleast some cells of the subject. Thus the invention provides methods ofreducing the vulnerability or propensity of a cell or subject to becomeinfected and/or to experience adverse effects due to a virus.“Resistance” to a virus typically refers to the ability to defendagainst infection. For purposes of description, the invention willmainly be described in terms of inhibiting virus infection. However, itwill be understood that, unless otherwise indicated, the inventivemethods of inhibiting virus infection of a cell or subject could bedescribed as inhibiting susceptibility of the cell or subject to virusinfection or increasing resistance of the cell or subject to virusinfection.

In some aspects, the invention provides methods of selecting atherapeutic agent for a subject, the method comprising (a) determiningwhether the subject is infected by a virus for which PLA2G16 is a hostcell factor; and (b) selecting a compound that inhibits PLA2G16 as atherapeutic agent for the subject if the subject is infected by a virusfor which PLA2S16 is a host cell factor. In some embodiments, the methodfurther comprises administering a compound that inhibits PLA2G16 to thesubject.

In some aspects, the invention provides methods of determining whether asubject is a candidate for treatment with a compound that inhibitsPLA2G16. In some embodiments, the method comprises determining whetherthe subject is infected by, or at risk of infection by, a virus forwhich PLA2G16 is a host cell factor, wherein if the subject is infectedby a virus for which PLA2G16 is a host cell factor, the subject is acandidate for treatment with a compound that inhibits PLA2G16. In someembodiments, the method comprises determining whether the subject isinfected by, or at risk of infection by, a picornavirus, wherein if thesubject is infected with a picornavirus, the subject is a candidate fortreatment with a compound that inhibits PLA2G16. In some embodiments,the method further comprises administering a compound that inhibitsPLA2G16 to the subject.

In some aspects, the invention provides methods of treating a subject inneed of treatment for a viral infection. In some embodiments, themethods comprise selecting a compound that inhibits PLA2G16 as atherapeutic agent for the subject. In some embodiments, the methodscomprise administering a compound that inhibits PLA2G16 to the subject.In some embodiments, the methods of treatment comprise providing asubject in need of treatment for a viral infection. In some embodiments,the methods of treatment comprise diagnosing a subject as being infectedwith a virus. The subject may have one or more symptoms or signs of aviral infection, e.g., one or more symptoms or signs associated with apathological state resulting from infection by a virus. In someembodiments, the method comprises administering a pharmaceuticalcomposition comprising the compound to the subject. “Administration” cancomprise direct administration or indirect administration. “Indirect”administration comprises activities such as providing, prescribing,directing another individual to administer, or in any way making acompound available to a subject.

III. Viruses and Viral Diseases

In some aspects, the invention relates to inhibiting infection of a cellor subject by a virus, wherein PLA2G16 promotes or plays a role in oneor more steps of the life cycle of the virus. In some embodiments, thevirus is capable of infecting cells of one or more animal species, e.g.,one or more vertebrate species, e.g., mammalian or avian species,wherein the cells express PLA2G16. In various embodiments, the inventionmay be applied to any virus whose capacity to infect a cell, e.g., ananimal cell, is reduced if PLA2G16 is inhibited. While the invention isdescribed herein mainly in reference to certain viruses of interest,embodiments of the invention can be applied to any virus whereinexpression of a PLA2G16 polypeptide in the cell promotes or plays a rolein one or more steps of the viral life cycle. In some embodiments, thevirus is of medical importance, e.g., it is recognized in the medicalart as a causative agent of one or more diseases that affect humans. Insome embodiments, the virus is of veterinary importance, e.g., it isrecognized in the veterinary art as a causative agent of one or morediseases that affect non-human animals. See, e.g., Knipe & Howley,supra; Büchen-Osmond, C. supra, and Virus Descriptions in “ICTVdB—TheUniversal Virus Database”, supra for discussion of various virusfamilies, including viruses of medical and/or veterinary importance.

In some embodiments, the virus has a single-stranded RNA (ssRNA) genome.In some embodiments, the ssRNA genome virus is positive stranded. Insome embodiments, the virus is a non-enveloped virus and/or has anicosahedral virion or nucleocapsid morphology. In some embodiments, thevirus is a member of the picornavirus-like superfamily (such viruses arealso termed “picorna-like viruses” herein). Viruses of thepicornavirus-like superfamily are positive-sense ssRNA viruses that arecharacterized by a partially conserved set of genes that consists of anRNA dependent RNA polymerase (RdRp), a chymotrypsin-like protease(3CPro), a superfamily 3 helicase (S3H) and a genome-linked protein(viral protein, genome linked, VPg). The picornavirus-like superfamilyencompasses the proposed order Picornavirales (discussed below) as wellas various virus genera and families falling outside the proposed order,including, e.g., Caliciviridae and Astroviridae. See, e.g., Koonin, E V,et al., Nature Reviews Microbiology, 6:925-939, 2008.

In some embodiments, the virus is a member of the proposed orderPicornavirales. This order includes viruses that infect eukaryotes andthat share the following properties: (i) a positive-sense RNA genome,usually with a 5′-bound VPg and 3′-polyadenylated, (ii) genometranslation into autoproteolytically processed polyprotein(s), (iii)capsid proteins organized in a module containing three relatedjelly-roll domains which form small icosahedral, non-enveloped particleswith a pseudo-T=3 symmetry, and (iv) a three-domain module containing asuperfamily III helicase, a (cysteine) proteinase with achymotrypsin-like fold and an RNA-dependent RNA polymerase. According tothese criteria, the order Picornavirales includes the familiesPicornaviridae, Comoviridae, Dicistroviridae, Marnaviridae, Sequiviridaeand the genera Cheravirus, Iflavirus and Sadwavirus. Other taxa of“picorna-like” viruses, e.g. Potyviridae, Caliciviridae, Hypoviridae, donot conform to several of the above criteria and are more remotelyrelated. The family Caliciviridae is composed of small (27-40 nm),nonenveloped, icosahedral viruses and include the four genera Norovirus,Sapovirus, Vesivirus, and Lagovirus. The major pathogens of medicalimportance are the noroviruses, which are a major cause of acutegastroenteritis. Important veterinary pathogens include vesirivursessuch as feline calicivirus (FCV) and rabbit hemorrhagic disease virus(RHDV). The family Astroviridae includes human and animal astrovirusesthat have icosahedral morphology and a characteristic starlike surfacestructure when viewed by electron microscopy. They are important agentsof gastroenteritis and diarrhea in humans as well as various animals,including mammals (e.g., pigs) and avians.

In some embodiments of particular interest, the invention relates toinhibiting infection by viruses that are members of the Picornaviridaefamily (also termed “picornaviruses” or “Picornaviruses” herein).Picornaviruses (like other members of the picornavirus-like superfamily)are nonenveloped viruses with a single-stranded genome of positivepolarity. They share a common genomic organization with a long 5′untranslated region (UTR) (e.g., at least about 500 nucleotides (nt) upto about 1200 nt long) containing an internal ribosome entry site(IRES), a single open reading frame (ORF) encoding a polyprotein that isproteolytically processed, and a short 3′ UTR followed by a polyA tail(Knipe & Howley, supra). Major distinguishing features among differentpicornaviruses include, among others, the secondary structure of the 5′UTR and IRES.

The picornavirus family includes twelve genera: Aphthovirus,Avihepatovirus, Cardiovirus, Enterovirus, Erbovirus, Hepatovirus,Kobovirus, Parechovirus, Sapelovirus, Senecavirus, Teschovirus, andTremovirus (see, “ICTVdB—The Universal Virus Database”, Virus Taxonomy:2009 Release v4, supra). A virus that is a members of one of thesegenera may be referred to as anaphthovirus, avihepatovirus, cardiovirus,enterovirus, erbovirus, hepatovirus, kobovirus, parechovirus,rhinoviruses, sapelovirus, senecavirus, teschovirus, or tremovirus,respectively These genera include numerous viruses that infectvertebrates, and a number of them contain members that are importantcauses of disease in humans and/or in non-human animals. For example,aphthoviruses include foot-and-mouth disease viruses, which infectcloven-footed animals such as cattle, goats, pigs, and sheep.Cardioviruses include two distinct clusters, the first of which includesencephalomyocarditisvirus and the second of which includes Theiler'smurine encephalomyelitis virus and related viruses, including some thatinfect humans.

Human enteroviruses are common causes of mild upper respiratory symptomsand flu-like illnesses, among others. Less commonly, they can result inmore serious conditions such as viral meningitis, myocarditis, orcentral nervous system conditions such as encephalitis. The Enterovirusgenus includes the following 10 species, as set forth by the ICTV in its2009 release (available athttp://ictvonline.org/virusTaxonomy.asp?version=2009): Human enterovirusA, Human enterovirus B, Human enterovirus C, Human enterovirus D, Simianenterovirus A, Bovine enterovirus, Porcine enterovirus B, Humanrhinovirus A, Human rhinovirus B and Human rhinovirus C. Many of thesespecies encompass multiple serotypes, which can in turn include multiplestrains. The Human enterovirus species collectively encompasspolioviruses, coxsackievirus, echoviruses, and numerous otherenteroviruses that infect humans. The Enterovirus genus also encompassesnumerous nonhuman enteric viruses. The poliovirus serotypes poliovirus(PV)-1, PV-2, and PV-3 are included within the Human enterovirus Cspecies. Although poliovirus has been largely eradicated throughwidespread use of effective vaccines, other viruses within theEnterovirus genus are frequent causes of acute and chronic humandiseases.

Coxsackieviruses are divided into group A and group B viruses based onearly observations of their pathogenicity in mice. Coxsackieviruses areassociated with a range of diseases in human including asepticmeningitis, hand-foot-mouth disease, herpangina, myocarditis (sometimesleading to cardiomyopathy), and pancreatitis, and may be an etiologicfactor in type I diabetes (See, e.g., articles in Curr Top MicrobiolImmunol. Vol. 323, 2008). Coxsackieviruses are classified among theHuman enterovirus A, Human enterovirus B, and Human enterovirus Cspecies. Exemplary coxsackieviruses include serotypes CV-A2, CV-A3,CV-A4, CV-A5, CV-A6, CV-A7, CV-A8, CV-A10, CV-A12, CV-A14, CV-A16,CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, CV-B6, CV-A9, CV-A1, CV-A11, CV-A13,CV-A17, CV-A19, CV-A20, CV-A21, CV-A22, CV-A24.

Human enterovirus A, Human enterovirus B, Human enterovirus C, and Humanenterovirus D species include additional enteroviruses such as serotypesEV-71, EV-76, EV-89, EV-90, EV-91, EV-92, EV-69, EV-73, EV-74, EV-75,EV-77, EV-78, EV-79, EV-80, EV-81, EV-82, EV-83, EV-84, EV-85, EV-86,EV-87, EV-88, EV-93, EV-97, EV-98, EV-100, EV-101, EV-106, EV-107,EV-95, EV-96, EV-99, EV-102, EV-104, EV-105, EV-109, EV-68, EV-70, andEV-94. For example, enterovirus 71 (EV-71) is a pathogenic enterovirusserotype that causes recurrent outbreaks in different parts of theworld. It can infect the central nervous system and may cause death andlong-term neurological sequelae in humans, especially infants and youngchildren (Lin, Y-W., et al., Journal of Virology, 83(13): 6477-6483,2009, and references therein). EV-71 may also cause diarrhea, rashes,and hand, foot and mouth disease.

Member of Human rhinovirus A and Human rhinovirus B species(“rhinoviruses”) replicate in the nasopharynx and sometimes in the lowerrespiratory tract. These viruses (of which more than 100 serotypesexist) are important etiologic agents of the common cold in humans andcan cause more severe disease as well, particularly in susceptibleindividuals.

The Teschovirus genus includes porcine teschovirus, which causespolioencephalitis in pigs. Hepatoviruses include human hepatitis A virus(HAV), which causes hepatitis A, an acute liver infection.

Additional picornaviruses continue to be discovered. See, e.g., Kapoor,A., et al., A highly prevalent and genetically diversifiedPicornaviridae genus in South Asian children. Proc Natl Acad Sci USA.105(51):20482-7, 2008, describing members of the proposed cosavirusgenus. More recently, a novel virus which has been designated asklassevirus was discovered using high throughput sequencing (see, e.g.,Greninger, A L, et al., The complete genome of klassevirus—a novelpicornavirus in pediatric stool, Virol J., 6:82-2009).

Those of skill in the art will appreciate that virus taxonomy andclassification continue to evolve and that viruses can be reclassified,e.g., as additional viruses are discovered or studied, e.g., as viralgenes are sequenced, and/or as relationships between viruses becomeevident. Thus certain viruses may have been reclassified by the ICTVsubsequent to publication of certain references cited herein and/or maybe reclassified in the future. Furthermore, those of skill in the artwill appreciate that many publications and references relating toviruses do not adhere to conventions established by the ICTV, may havepreceded the establishment of these conventions, and/or may employformal and/or informal vernacular nomenclature. Identifyingcharacteristics of viruses (and strains and variants thereof) are wellestablished and known in the art. Characterized reference samples ofnumerous viruses are deposited in and typically available from variousinternationally recognized biological resource centers or culturecollections such as the American Type Culture Collection (ATCC)(Manassas, Va.; http://www.atcc.org/), National Collection of PathogenicViruses (NCPV) of the Health Protection Agency Culture Collections ofthe Health Protection Agency of the United Kingdom (Porton DownmSalisbury UK; http://www.hpacultures.org.uk/aboutus/ncpv.jsp) and/orinternationally recognized specialty groups, as are reagents of use toidentify and/or characterize viruses. Characterization and/orclassification can be based on properties such as nucleic acid and/orpolypeptide sequences, reactivity with immunological reagents (e.g.,antisera), etc. Genome sequences of numerous enteroviruses, includingthose of numerous human enteroviruses, are publicly available, e.g., onthe website of the European Bioinformatics Institute(http://www.ebi.ac.uk/), National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/), and in the scientific literature.

Picornavirus structure and life cycle have been extensively studied(see, e.g., Knipe & Howley, supra). Briefly, the picornavirus capsid istypically composed of four structural proteins: VP1, VP2, VP3 and VP4.(Parechoviruses contain only VP1, VP2, and VP0, the uncleaved precursorof VP2+VP4). The basic building block of the picornavirus capsid, termedthe protomer, contains one copy of VP1, VP2, VP3, and VP4. VP1, VP2, andVP3 form a shell with VP4 on its inner surface. Differences in the aminoacid sequences of certain portions of VP 1, VP2, and VP3 give differentpicornaviruses distinct morphologies and antigenicities.

Replication of picornaviruses occurs in the cell cytoplasm.Picornaviruses initiate infection by attaching to a receptor on the hostcell membrane, which is followed by uncoating and entry of the viralgenome into the cytoplasm. The poliovirus receptor (PVR, CD155) and themajor group rhinovirus (ICAM-1) were identified in 1989, and since thattime receptors for a number of other picornaviruses have beenidentified. Some picornaviruses typically require co-receptors forinfection. For example, many enteroviruses bind to decay-acceleratingfactor (DAF; CD55) but infection typically requires presence of anadditional molecule, e.g., ICAM-1 or an integrin family member. The RNAgenome is translated on entry into the cytoplasm to a single polyproteinthat is cleaved during translation by virus-encoded proteases (mainly2Apro and C3pro or 3CDpro) to produce all the viral proteins needed forviral replication. Some of the uncleaved precursors also have functionsduring viral replication. Among the viral proteins synthesized are theviral RNA-dependent RNA polymerase and accessory proteins required forgenome replication and mRNA synthesis. The first step of genomereplication is copying of the positive-stranded RNA to generate anegative-stranded intermediate, which is used as a template forsynthesis of additional positive strands. Encapsidation begins oncesufficient capsid proteins have accumulated.

Many picornaviruses produce characteristic morphologic changes termed“cytopathic effects” in infected cells. Cytopathic effects can includechromatin condensation, nuclear blebbing, proliferation of membranousvesicles, leakage of intracellular contents, and shriveling of theentire cell. In the case of many picornavirus species, virions arereleased from infected cells as a consequence of cell lysis. Otherpicornaviruses (e.g., hepatitis A virus) are released from cells in theabsence of cell lysis. In some embodiments of the invention, cytopathiceffect(s) and/or virion release is assessed to determine whether a cellor subject is infected with a virus. In some embodiments, cytopathiceffect(s) and/or virion release is assessed to determine whether acompound inhibits a virus infection.

IV. PLA2G16 Polypeptides

PLA2G16 is an ˜18 kilodalton protein that is highly expressed invertebrate adipose tissue (especially white adipose tissue) and is alsoexpressed at lower levels in a wide variety of vertebrate tissues andcultured cell lines. PLA2G16 is also known as adipose-specificphospholipase A2 (AdPLA), HRAS-like suppressor 3 (HRASLA3), and byseveral other names. One of skill in the art will readily be able toobtain PLA2G16 genomic and mRNA sequences and the PLA2G16 protein frompublicly available databases. The human gene encoding PLA2G16 has beenassigned GeneID: 11145 in the Gene database of the National Center forBiotechnology Information (NCBI; www.ncbi.nlm.nih.gov). Genes encodingPLA2G16 from mouse and rat have been assigned the following Gene IDs:Gene ID: 225845 (Mus musculus); Gene ID: 24913 (Rattus norvegicus). Oneof skill in the art will readily be able to obtain the sequences ofPLA2G16 mRNA and protein from these and other species. For example,accession numbers for the human PLA2G16 mRNA and protein ReferenceSequences available at the NCBI are as follows: NM_(—)001128203(transcript variant 2) and NP_(—)001121675 (protein). NM_(—)007069.3(transcript variant 1) and NP_(—)009000.2 (protein). Transcript variant1 represents the longer transcript. Variants 1 and 2 encode the sameisoform but differ in the 5′ untranslated region (UTR).

PLA2G16 has phospholipase activity and significantly lower butdetectable lysophospholipase activity (Duncan, R E, et al., J BiolChem., 283(37):25428-36, 2008). PLA₂ proteins are enzymes that catalyzehydrolysis of the sn-2 bond of phospholipids (Schaloske, R H and Dennis,E A, Biochim. Biophys. Acta 1761, 1246-1259, 2006). PLA2G16 was shown togenerate free fatty acid and lysophospholipid from phosphatidylcholinewith a preference for hydrolysis at the sn-2 position, suggesting thatthe protein is a PLA₂ (Duncan, supra). PLA2G16 was found in associationwith intracellular membranes and has a C-terminal presumed membranespanning domain whose deletion caused a loss of activity. Mutationalanalysis showed that certain highly conserved amino acids, includingHis-23, Cys-113, Gln-129 and Asn-112, were essential for catalysis, butthat mutation of Asp-30 or His-80 to alanine had no effect. Thus PLA2G16appears to contain His and Cys active catalytic residues rather than aHis/Asp catalytic diad or a Ser/His/Asp catalytic triad as found in someother PLA₂s. Calcium was found to activate PLA2G16 but is not essentialfor activity. Since PLA2G16 does not fit clearly into any of thepreviously identified 15 major groups of PLA₂ it was proposed to be thefirst member of a distinct group of calcium-dependent phospholipase A₂s(Group XVI) (Duncan, supra).

PLA2G16 is also known in the art as adipocyte phospholipase A2 (AdPLA)(Jaworski, K., et al. Nat Med., 15(2):159-68, 2009). It is the majorPLA₂ in adipose tissue and plays an important role in regulatingadipocyte lipolysis. PLA2G16 null mice were viable and had normal weightat weaning but gained weight more slowly than wild-type littermatesdespite having equivalent food intakes (Jaworski, K., supra). Bystandard pathology analysis PLA2G16 null mice showed no evidence of anygross, microscopic, or functional abnormalities, aside from reducedadiposity. Blood cell profile and immunological parameters in serum andadipose tissue were not changed in these mice compared to wild-typemice. These results suggest that methods of the present invention thatcomprise inhibiting PLA2G16 in order to inhibit viral infection arelikely to be well tolerated in isolated cells and in subjects ofinterest, e.g., humans and other vertebrates.

In some embodiments, a “PLA2G16 polypeptide” is a polypeptide whosesequence comprises or consists of the sequence of a PLA2G16 polypeptideof a multicellular organism (e.g., a vertebrate, e.g., a mammal, such asa human, mouse, rat, bovine, etc.). A naturally occurring PLA2G16polypeptide or a polypeptide identical in sequence to a naturallyoccurring PLA2G16 polypeptide is referred to as a “native PLA2G16polypeptide” or simply “PLA2G16” herein. Exemplary native PLA2G16polypeptides are depicted in FIG. 8 and under the accession numbersmentioned above. In some embodiments, a PLA2G16 polypeptide is a variantof PLA2G16 (“PLA2G16 variant”). PLA2G16 variants include polypeptidesthat differ by one or more amino acid substitutions, additions, ordeletions, relative to PLA2G16. An addition can be an insertion withinthe polypeptide or an addition at the N- or C-terminus. In someembodiments, the number of amino acids substituted, deleted, or addedcan be for example, about 1 to 30, e.g., about 1 to 20, e.g., about 1 to10, e.g., about 1 to 5, e.g., 1, 2, 3, 4, or 5. In some embodiments, aPLA2G16 variant comprises a polypeptide whose sequence is homologous tothe sequence of PLA2G16 over at least 50 amino acids, at least 100 aminoacids, at least 150 amino acids, or over the full length of PLA2G16 (butis not identical in sequence to native PLA2G16). In some embodiments, aPLA2G16 variant comprises a polypeptide at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or more identical to PLA2G16 (e.g., fromhuman, mouse, rat, dog, cow) over at least 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% of PLA2G16. In some embodiments, aPLA2G16 variant comprises a polypeptide at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or more identical to at least amino acids 23-113 ofhuman or mouse PLA2G16. In some embodiments, a PLA2G16 polypeptidecomprises a polypeptide at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or more identical to at least amino acids 23-129 of human or mousePLA2G16.

In some embodiments, a PLA2G16 polypeptide comprises or consists of aPLA2G16 fragment. A PLA2G16 fragment is a polypeptide that is shorterthan PLA2G16 and is identical to PLA2G16 over the length of the shorterpolypeptide. In some embodiments, a PLA2G16 fragment is at least 50%,60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% as long as nativePLA2G16. In some embodiments, a fragment consists of amino acids 23-113or 23-129 of human or mouse PLA2G16. In some embodiments, one or moreamino acids at the C-terminus are deleted. For example, in someembodiments at least the membrane spanning domain at the C-terminus isdeleted. For example, in some embodiments, at least the C-terminal 30amino acids are deleted. In some embodiments, one or more amino acids atthe N-terminus are deleted.

In some embodiments, a PLA2G16 polypeptide comprises a heterologouspolypeptide portion. The heterologous portion often has a sequence thatis not present in or homologous to native PLA2G16. A heterologousportion may be, e.g., between 5 and about 5,000 amino acids long, orlonger. Often it is between 5 and about 1,000 amino acids long. In someembodiments, a heterologous portion comprises a sequence that is foundin a different polypeptide, e.g., a functional domain. In someembodiments, a heterologous portion comprises a sequence useful forpurifying, expressing, solubilizing, and/or detecting the polypeptide.In some embodiments, a heterologous portion comprises a polypeptide“tag”, e.g., an affinity tag or epitope tag. For example, the tag can bean affinity tag (e.g., HA, TAP, Myc, 6×His, Flag, GST), fluorescent orluminescent protein (e.g., EGFP, ECFP, EYFP, Cerulean, DsRed, mCherry),solubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, or amonomeric mutant of the Ocr protein of bacteriophage T7). See, e.g.,Esposito D and Chatterjee D K. Curr Opin Biotechnol.; 17(4):353-8(2006). In some embodiments, a tag can serve multiple functions. A tagis often relatively small, e.g., ranging from a few amino acids up toabout 100 amino acids long. In some embodiments a tag is more than 100amino acids long, e.g., up to about 500 amino acids long, or more. Insome embodiments, a PLA2G16 polypeptide has a tag located at the N- orC-terminus, e.g., as an N- or C-terminal fusion. The polypeptide couldcomprise multiple tags. In some embodiments, a 6×His tag and a NUS tagare present, e.g., at the N-terminus. In some embodiments, a tag iscleavable, so that it can be removed from the polypeptide, e.g., by aprotease. In some embodiments, this is achieved by including a sequenceencoding a protease cleavage site between the sequence encoding theportion homologous to PLA2G16 and the tag. Exemplary proteases include,e.g., thrombin, TEV protease, Factor Xa, PreScission protease, etc. Insome embodiments, a “self-cleaving” tag is used. See, e.g.,PCT/US05/05763. Sequences encoding a tag can be located 5′ or 3′ withrespect to a polynucleotide encoding the polypeptide (or both). In someembodiments a tag or other heterologous sequence is separated from therest of the polypeptide by a polypeptide linker. For example, a linkercan be a short polypeptide (e.g., 15-25 amino acids). Often a linker iscomposed of small amino acid residues such as serine, glycine, and/oralanine. A heterologous domain could comprise a transmembrane domain, asecretion signal domain, etc.

In some embodiments, a PLA2G16 variant is a functional variant, i.e.,the variant at least in part retains at least one biological activity ofPLA2G16. In some embodiments, a functional variant retains sufficientactivity to be distinguishable from a non-homologous protein orcatalytically inactive PLA2G16 polypeptide (e.g., a PLA2G16 polypeptidehaving a C113A substitution) when used in an assay of the presentinvention. In some embodiments, the activity is phospholipase A2activity, e.g., as measured by ability to catalyze hydrolysis of thesn-2 bond of phospholipids. In some embodiments, the activity islysophospholipase activity. In some embodiments, a PLA2G16 variantretains the ability of native PLA2G16 to serve as a host cell factor fora virus. For example, the PLA2G16 variant has sufficient activity sothat expressing it in a vertebrate cell that is resistant to viralinfection because the cell's PLA2G16 gene is disabled (e.g., by a genetrap insertion) renders the cell sensitive to viral infection. In someembodiments, a functional PLA2G16 variant retains at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more ofthe activity of PLA2G16, e.g., about equal activity. In someembodiments, a functional variant may have greater activity thanPLA2G16.

One of skill in the art can readily generate functional PLA2G16 variantsor fragments. As discussed above, considerable information is availableregarding PLA2G16, including identification of various residuesimportant for activity and various residues that may be altered withoutsignificantly decreasing activity, as well as alignments with other PLA2polypeptides (see, e.g., Duncan, et al, supra). In some embodiments, aPLA2G16 variant comprises one or more conservative amino acidsubstitutions relative to PLA2G16. Conservative substitutions may bemade on the basis of similarity in side chain size, polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues involved. As known in the art, such substitutions are,in general, more likely to result in a variant that retains activity ascompared with non-conservative substitutions. In one embodiment, aminoacids are classified as follows:

Special: C

Neutral and small: A, G, P, S, TPolar and relatively small: N, D, Q, EPolar and relatively large: R, H, KNonpolar and relatively small: I, L, M, VNonpolar and relatively large: F, W, Y

Special: C

See, e.g., Zhang, J. J. Mol. Evol. 50:56-68, 2000). In some embodiments,proline (P) is considered to be in its own group as a second specialamino acid. Within a particular group, certain substitutions may be ofparticular interest, e.g., replacements of leucine by isoleucine (orvice versa), serine by threonine (or vice versa), or alanine by glycine(or vice versa). Of course non-conservative substitutions are oftencompatible with retaining function as well. In some embodiments, asubstitution or deletion does not alter or delete an amino acidimportant for activity, e.g., amino acid His-23, Cys-113, Gln-129 orAsn-112. In some embodiments, a deletion does not remove all or asubstantial portion of the C-terminal 36 amino acids. For example, insome embodiments, a deletion does not remove the transmembrane domain.In some embodiments, an alteration is at an amino acid that differsamong PLA2G16 of different species. In some embodiments, a substitutionalters an amino acid to that present at a corresponding position in adifferent species. In some embodiments, a functional PLA2G16 variantcomprises a polypeptide at least 95%, 96%, 97%, 98%, 99% or 100%identical to PLA2G16, e.g., over at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% or 100% of the full length of PLA2G16. In someembodiments, a functional PLA2G16 variant comprises a polypeptide atleast 95%, 96%, 97%, 98%, 99% or 100% identical to PLA2G16 e.g., over atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of thefull length of PLA2G16, and comprises a tag at the N- and/or C-terminus.PLA2G16 variants could be tested in cell-free and/or cell-based assaysto assess their activity.

In some embodiments, a variant or fragment of PLA2G16 that hassubstantially reduced activity as compared with the activity of nativePLA2G16 (e.g., less than 10% of the activity of native PLA2G16) isuseful as a PLA2G16 inhibitor or antiviral compound. For example, suchpolypeptide could interfere with the function of native PLA2G16 in viralinfection, e.g., by competing with native PLA2G16. In some embodiments,a variant or fragment of PLA2G16 that has substantially reduced activityas compared with the activity of native PLA2G16 is useful a control oras an immunogen or for crystallization or binding studies.

A PLA2G16 polypeptide, e.g., a native PLA2G16 polypeptide or a PLA2G16variant can be produced using standard recombinant DNA techniques. Anucleic acid encoding PLA2G16 can readily be obtained, e.g., from cellsthat express PLA2G16 (e.g., by PCR or other amplification methods or bycloning) or by synthesis based on a known PLA2G16 cDNA or polypeptidesequence. One of skill in the art would know that due to the degeneracyof the genetic code, numerous different nucleic acid sequences wouldencode the desired polypeptide. Optionally, a sequence iscodon-optimized for expression in a host cell of choice. A nucleic thatencodes a PLA2G16 variant can readily be generated, e.g., by modifyingnative PLA2G16 using, e.g., site-directed mutagenesis, or by otherstandard methods.

A nucleic acid encoding the desired polypeptide, operably linked toappropriate expression control elements, usually in a vector such as aplasmid or virus (e.g., as part of the viral genome), is introduced intoprokaryotic or eukaryotic cells. In other embodiments, a PLA2G16polypeptide is produced using in vitro translation. Exemplary cellsinclude, e.g., bacterial cells (e.g., E. coli), insect cells, mammaliancells, plant cells, fungal cells (e.g., yeast). One of skill in the artwill be aware of suitable expression control elements (e.g., promoters).Promoters may be constitutive or regulatable, e.g., inducible orrepressible. Exemplary promoters suitable for use in bacterial cellsinclude, e.g., Lac, Trp, Tac, araBAD (e.g., in a pBAD vectors), phagepromoters such as T7 or T3. Exemplary expression control sequencesuseful for directing expression in mammalian cells include, e.g., theearly and late promoters of SV40, adenovirus or cytomegalovirusimmediate early promoter, or viral promoter/enhancer sequences,retroviral LTRs, promoters or promoter/enhancers from mammalian genes,e.g., actin, EF-1 alpha, metallothionein, etc. The polyhedrin promoterof the baculovirus system is of use to express proteins in insect cells.One of skill in the art will be aware of numerous expression vectorsthat contain appropriate expression control element(s), selectablemarkers, cloning sites, etc., and can be conveniently used to express apolypeptide of interest. Optionally, such vectors include sequencesencoding a tag, to allow convenient production of a polypeptidecomprising a tag. Suitable methods for introducing vectors intobacteria, yeast, plant, or animal cells (e.g., transformation,transfection, infection, electroporation, etc.), and, if desired,selecting cells that have taken up the vector and deriving stable celllines. Transgenic animals or plants that express the polypeptide couldbe produced using methods known in the art.

To produce a PLA2G16 polypeptide, cells are maintained in culture for asuitable time period, and the polypeptide is isolated and optionallyfurther purified. (Of course a PLA2G16 polypeptide could also beisolated from cells or tissues obtained directly from an organism thatexpresses it.) Standard protein isolation/purification techniques can beused. In some embodiments, affinity-based methods are used. For example,an antibody to PLA2G16 can be employed. In the case of tagged PLA2G16polypeptides, an appropriate isolation method can be selected dependingon the particular tag used.

V. Compositions and Methods for Inhibiting PLA2G16

The term “PLA2G16 inhibitor” refers to a compound that inhibits PLA2G16expression and/or inhibits one or more activities of PLA2G16. Forexample, a compound is “PLA2G16 inhibitor” if one or more PLA2G16activities is reduced in the presence of the compound as compared withits absence and/or if the level or amount of PLA2G16 protein or geneproduct is reduced in the presence of the compound as compared with itsabsence. In certain embodiments, PLA2G16 inhibitors act directly onPLA2G16 in the sense that they physically interact with PLA2G16. Inother embodiments, inhibitors act indirectly on PLA2G16. A PLA2G16inhibitor can be, e.g., a small molecule, nucleic acid, oligonucleotide,polypeptide, peptide, lipid, phospholipid, etc. In some embodiments, aPLA2G16 inhibitor is an RNAi agent, antisense oligonucleotide, aptamer,or antibody. In some embodiments, a PLA2G16 inhibitor is a smallmolecule.

The invention provides a number of different methods of inhibitingPLA2G16. As used herein, methods of inhibiting PLA2G16 encompass methodsthat result in a decreased amount of PLA2G16 polypeptide and methodsthat interfere with PLA2G16 molecular function. In some embodiments,PLA2G16 is inhibited by inhibiting or interfering with PLA2G16expression, so that a decreased amount of PLA2G16 polypeptide isproduced. As used herein, “expression” encompasses the cellularprocesses involved in producing a polypeptide and include transcription,mRNA processing and transport (in the case of eukaryotic cells), andmRNA translation. A variety of methods useful for inhibiting orinterfering with expression can be applied in embodiments of the presentinvention. In general, such methods result in decreased synthesis ofPLA2G16 polypeptide and as a result, a reduction in the total level ofPLA2G16 molecular functional activity present.

In some embodiments, PLA2G16 expression is inhibited using RNAinterference (RNAi). Exemplary sequences for RNAi agents (e.g., siRNAs)that inhibit PLA2G16 expression are provided in the Examples. Additionalsequences can be selected using various approaches known in the artincluding. If desired, such sequences can be selected to minimize“off-target” effects. In some embodiments, position-specific chemicalmodification is used to reduce potential off-target effects. In someembodiments, at least two different siRNAs targeted to the PLA2G16 geneare used (e.g., in combination). RNAi is use of herein for a variety ofpurposes. For example, an RNAi agent can be used as a PLA2G16 inhibitor,e.g., for therapeutic or research purposes. An RNAi agent that inhibitsPLA2G16 can be useful to confirm that the effect of a second compound,e.g., a small molecule, is due to an effect on PLA2G16 (rather than onanother protein). For example, a small molecule that is a putativespecific inhibitor of PLA2G16 may be expected not to have an effect in acell in which PLA2G16 expression is inhibited by RNAi. In other aspects,RNAi is used to inhibit expression of a PLA2 other than PLA2G16, whichmay be expressed by a cell. Inhibiting other PLA2 enzymes may facilitateidentification of compounds that inhibit PLA2G16.

In some embodiments of the invention, PLA2G16 expression is inhibitedusing an antisense approach in which one or more oligonucleotidescomplementary to mRNA encoding PLA2G16 is delivered to cells andhybridizes to the PLA2G16 mRNA resulting in, e.g., degradation of themRNA by RNase H or blockage of translation by steric hindrance.

In some embodiments of the invention, a PLA2G16 inhibitor inhibits atleast one molecular function of PLA2G16. In some embodiments, themolecular function is a catalytic activity, e.g., phospholipase activityand/or lysophospholipase activity. For example, the activity may bephospholipase A2 activity, i.e., ability to catalyze hydrolysis of thesn-2 bond of phospholipids. In some embodiments, a compound directlyinhibits a molecular function of PLA2G16. “Direct inhibition” refers toa physical interaction (binding) with a target that inhibits a molecularfunction of the target. For example, binding of a PLA2G16 inhibitor toPLA2G16 can interfere with the enzyme's ability to catalyze a reactionand/or prevent a substrate from entering the active site. A variety ofcompounds can be used to directly inhibit PLA2G16 molecular function.Exemplary compounds that directly inhibit PLA2G16 can be, e.g., smallmolecules, antibodies, or aptamers. In some embodiments, a directinhibitor is a substrate analog (e.g., a phospholipid analog) or atransition state analog.

In some embodiments, an inhibitor is an irreversible inhibitor. Mostirreversible enzyme inhibitors react with the enzyme and change itchemically, such as by modifying amino acid residue(s) that are neededfor enzymatic activity. For example, an irreversible inhibitor cancomprise one or more reactive functional groups such as an aldehyde,haloalkane, alkene, fluorophosphonate (e.g., alkyl fluorophosphonate),Michael acceptor, phenyl sulfonate, methylketone, e.g., a halogenatedmethylketone or diazomethylketone, fluorophosphonate, vinyl ester, vinylsulfone, or vinyl sulfonamide. In some embodiments, an irreversiblePLA2G16 inhibitor comprises an electrophilic group that reacts with anamino acid side chain of PLA2G16. For example, the electrophilic groupmay react with an amino acid side chain containing a nucleophile such asa hydroxyl or sulfhydryl group. For example, the amino acid may becysteine, serine, or threonine. In another embodiment, an irreversibleinhibitor reacts with a histidine. Moieties sometimes referred to in theart as “cysteine traps” may be used in various embodiments. In someembodiments a cysteine-reactive moiety is a maleimide, isothiazolinone,tetrazole, lactam, or carbamate. A reactive functional group can beincorporated into a substrate analog or other molecule compatible withbinding to the enzyme, e.g., in or near the active site.

In other embodiments, a PLA2G16 inhibitor is a reversible inhibitor.Reversible inhibitors bind non-covalently and may bind to the enzyme,the enzyme-substrate complex, or both. Inhibition by a reversibleinhibitor may be classified as competitive inhibition, uncompetitiveinhibition, mixed inhibition, non-competitive inhibition. See, e.g.,Berg J. M, et al., Biochemistry, 6^(th) ed., W. H. Freeman and Company,2007. In some embodiments, a reversible inhibitor binds to the PLA2G16active site and/or competes with substrate(s) for access to the PLA2G16active site. In some embodiments a reversible inhibitor is anon-hydrolyzable substrate analog.

In some embodiments, the PLA2G16 inhibitor is an analog of a fatty acid,wherein the analog comprises an alkyl chain between 4 and about 30carbons long, e.g., between 12 and 20 carbons long. In some embodiments,the alkyl group is saturated. In some embodiments, the alkyl group isunsaturated. In some embodiments, the alkyl group is unbranched. In someembodiments, the alkyl group has the structure of an alkyl groupnaturally found in a fatty acid present in vertebrate cells. Exemplaryfatty acids include, e.g., myristoleic acid, palmitoleic acid, sapienicacid, oleic acid, linoleic acid, linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid, docosahexaenoic acid, lauric acid,myristic acid, palmitic acid, stearic acid, and eicosanoic acid. In someembodiments, the PLA2G16 inhibitor is an analog of arachidonic acid orlinoleic acid. In some embodiments, the analog is a methylated fattyacid. In some embodiments, the arachidonic acid analog is aneicosadienoic acid, such as 7,7-dimethyl-5,8-eicosadienoic acid.

In some embodiments, the inhibitor comprises an analog of a fatty acid,wherein the analog comprises a reactive functional group. In someembodiments, the fatty acid analog comprises a halogenated methyl ketonegroup instead of a carboxyl group. For example, the halogen can bechlorine, fluorine, bromine, or iodine in various embodiments. In someembodiments, the halogenated methyl ketone group is fluoromethyl ketone,trifluoromethyl ketone, or chloromethyl ketone. In some embodiments, thefatty acid is arachidonic acid. In one embodiment, the inhibitor is atrifluoromethyl ketone analog of arachidonic acid in which the COOHgroup is replaced with COCF3, i.e., the compound arachidonyltrifluoromethyl ketone (AACOCF3). AACOCF3 inhibits PLA2G16 and alsoinhibits cPLA₂ and sPLA₂ (Duncan, supra). It is believed that AACOCF3binds in a hydrophobic pocket of cPLA₂, and that the carbonyl group ofAACOCF3 forms a covalent bond with serine 228 in the active site (Streetet al., 1993; Trimble et al., 1993). Without wishing to be bound bytheory, AACOCF3 may react with the active site serine of PLA2G16. Inanother embodiment, the PLA2G16 inhibitor is methyl arachidonylfluorophosphate (MAFP) or an analog thereof (see, e.g., Martin, B R, etal., J. Pharm. Exp. Ther., 294 (3), 294:1209-1218, 2000). MAFP isbelieved to inhibit serine and cysteine hydrolases by covalently bindingto the enzyme. It inhibits PLA2G16 as well as iPLA2 and cPLA2, but notsPLA2 (Duncan, supra). Without wishing to be bound by theory, PLA2G16may require an active cysteine residue (i.e. Cys-113) that isinactivated by MAFP.

A variety of other compounds that inhibit one or more Group I-XV PLA2shave been identified. For example, U.S. Pat. Pub. No. 20080319065discloses compounds that contain a 2-oxoamide with a hydrocarbon tailand a four carbon tether and are reported to inhibit PLA2 Group IVA cPLA2 and/or Group VIA iPLA2 and/or Group V sPLA2. U.S. Pat. Pub. Nos.20030144282 and 20100029645 disclose inhibitors of various PLA2 enzymes.Other compounds that inhibit one or more PLA2 enzymes includepiperazines (see, e.g., WO2003048139); pyrimidone, pyridone, pyridinone,and pyrimidinone compounds (see, e.g., WO2002030904; WO 2001060805; WO2000027824; WO 2003087088; WO 2003086400; WO 2003/042218; WO2003042206;WO2002030911; WO2003041712), pyrrolidine derivatives (see, e.g.,WO1998033797). Without wishing to be bound by any theory, at least someof the compounds that inhibit one or more Group I-XV PLA2 enzymes mayalso inhibit PLA2G16. In some embodiments, the main mechanism of actionagainst such other Group I-XV PLA₂ does not involve specifically bindingto a sequence motif that is present in such other PLA2 but is absent inPLA2G16. For example, in some embodiments the compound is not whose mainmechanism of action involves binding to a GXSXG consensus motif or aCCXXHDXC motif.

In some embodiments, a PLA2G16 inhibitor comprises a peptide, e.g., apeptide identified using a display technique, such as phage display orribosome display. In some embodiments, a peptide comprises one or morenon-standard amino acids. In some embodiments, a peptide is cyclic. Forexample, the peptide can be cyclized via a disulfide bond or covalentlinkage, e.g., between the N- and C-terminal amino acids, between the N-or C-terminal amino acid an internal amino acid, or between two internalamino acids.

In some embodiments, a PLA2G16 inhibitor comprises an aptamer. Ingeneral, an aptamer is an often single-stranded oligonucleotide (e.g.,DNA or RNA, optionally containing one or more non-standard nucleotidesor modifications such as 2′-fluoro, 2′-amino, and/or 2′-methoxynucleotides) that binds to a particular molecule of interest. Aptamersare typically derived from an in vitro evolution and selection processsuch as SELEX. Methods for obtaining aptamers specific for a protein ofinterest are known in the art. See, e.g., Brody E N, Gold L. JBiotechnol., 74(1):5-13, 2000.

In some embodiments, a PLA2G16 inhibitor comprises an antibody orportion thereof. In some embodiments, the antibody is a single-chainantibody, diabody, triabody, or minibody. Standard methods of antibodyproduction known in the art can be used to produce an antibody, e.g., amonoclonal antibody, that binds to PLA2G16. In some embodiments, ananimal, e.g., a mouse or rabbit is immunized with PLA2G16 or a portionthereof, antibody producing cells are isolated, and a monoclonalantibody is identified using hybridoma technology. In some embodiments,the mouse is a transgenic mouse comprising at least some unrearrangedhuman immunoglobulin gene sequences and that preferably have a targeteddisruption of endogenous heavy and light chain murine sequences. In someembodiments, an antibody is identified or produced using recombinantnucleic acid technology (e.g., phage or yeast display). See, e.g.,Lonberg N. Fully human antibodies from transgenic mouse and phagedisplay platforms. Curr Opin Immunol. 20(4):450-9, 2008.

In some embodiments of the invention, a compound indirectly inhibitsPLA2G16. “Indirect inhibition” refers to inhibition of a target (e.g.,PLA2G16) by a mechanism that does not require physical interactionbetween the compound and the target. For example, the compound couldinhibit expression or activity of a polypeptide that is involved inlocalization or post-translational modification of PLA2G16, wherein suchlocalization or post-translational modification is important for PLA2G16molecular function.

In some embodiments, a PLA2G16 inhibitor is not a compound that is knownor suggested in the art to have antiviral activity and/or to be usefulin treating a subject in need of treatment for a viral infection. Insome embodiments, a PLA2G16 inhibitor is a compound that is known orsuggested in the art to have antiviral activity and/or to be useful intreating a subject in need of treatment for a viral infection but,optionally, may be administered or otherwise used in the presentinvention (i) to inhibit infection by a different virus, e.g., a virusagainst which the compound is not known or suggested to have antiviralactivity; (ii) in a different (e.g., more highly purified) form, in adifferent amount or composition, or in combination with one or moredifferent substances; (iii) by a different route or to a subject of adifferent species; and/or (iv) explicitly excluded from any one or moreof the inventive compositions and/or methods.

VI. Compositions and Methods for Identifying and/or Testing Compounds

The invention provides methods of identifying compounds useful forinhibiting viral infection and assay systems for performing theinventive methods. In some aspects, the invention provides a method ofdetermining whether a test compound is a candidate antiviral compound,the method comprising the step determining whether the test compoundinhibits PLA2G16 polypeptide, wherein if the compound inhibits PLA2G16,the compound is a candidate antiviral compound. In a related aspect, theinvention provides a method of identifying a candidate antiviralcompound comprising steps of: (a) providing a test compound; (b)determining whether the test compound inhibits PLA2G16, wherein if thecompound inhibits PLA2G16, the compound is a candidate antiviralcompound.

In some embodiments, the method comprises determining whether the testcompound inhibits expression of PLA2G16, wherein if the compoundinhibits PLA2G16 expression the compound is a candidate antiviralcompound. In some embodiments, the method comprises determining whetherthe compound inhibits a molecular function of PLA2G16, wherein if thecompound inhibits a molecular function of PLA2G16 the compound is acandidate antiviral compound. In some embodiments, the molecularfunction is an enzymatic activity, e.g., phospholipase A2 activity orlysophospholipase activity.

In some embodiments, a method is performed using a PLA2G16 polypeptideidentical in sequence to PLA2G16 that is naturally expressed by amulticellular organism, i.e., a native PLA2G16. In some embodiments, amethod is performed using a functional PLA2G16 variant. In someembodiments, the functional variant used in an inventive assay retainsat least 20%, 30%, 40%, 50%; 60%. 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or more of the phospholipase A2 activity of PLA2G16. In someembodiments, a functional variant retains at least 20%, 30%, 40%, 50%,60%. 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of thelysophospholipase activity of PLA2G16. In some embodiments, thefunctional variant retains at least 50% or at least 75% or has about thesame phospholipase A2 activity and/or lysophospholipase activity asnative PLA2G16. A PLA2G16 variant may have properties that make itconvenient to use in an inventive screening method, such as the presenceof a tag that facilitates production or purification of the protein. Acompound identified as an inhibitor using a PLA2G16 variant can befurther tested using native PLA2G16 to confirm its ability to inhibitthe native polypeptide.

A wide variety of test compounds can be used in the inventive methods.For example, a test compound can be a small molecule, polypeptide,peptide, nucleic acid, oligonucleotide, lipid, carbohydrate, or hybridmolecule. Compounds can be obtained from natural sources or producedsynthetically. Compounds can be at least partially pure or may bepresent in extracts or other types of mixtures. Extracts or fractionsthereof can be produced from, e.g., plants, animals, microorganisms,marine organisms, fermentation broths (e.g., soil, bacterial or fungalfermentation broths), etc. In some embodiments, a compound collection(“library”) is tested. The library may comprise, e.g., between 100 and500,000 compounds, or more. Compounds are often arrayed in multwellplates. They can be dissolved in a solvent (e.g., DMSO) or provided indry form, e.g., as a powder or solid. Collections of synthetic,semi-synthetic, and/or naturally occurring compounds can be tested.Compound libraries can comprise structurally related, structurallydiverse, or structurally unrelated compounds. Compounds may beartificial (having a structure invented by man and not found in nature)or naturally occurring. In some embodiments, a library comprises atleast some compounds that have been identified as “hits” or “leads” inother drug discovery programs and/or derivatives thereof. A compoundlibrary can comprise natural products and/or compounds generated usingnon-directed or directed synthetic organic chemistry. Often a compoundlibrary is a small molecule library. Other libraries of interest includepeptide or peptoid libraries, cDNA libraries, and oligonucleotidelibraries.

A library can be focused (e.g., composed primarily of compounds havingthe same core structure, derived from the same precursor, or having atleast one biochemical activity in common). In some embodiments,compounds that have been identified as inhibitors of one or more GroupI-XV PLA2 enzymes are tested. In some embodiments, the IC50 of acompound identified as a PLA2G16 inhibitor may be about 2, 5, 10, 20,50, 100, 250, 500, or 1000-fold lower for PLA2G16 versus one or moreother PLA2 enzymes (e.g., one, more than one, or all other PLA2 enzymespresent in humans and known to date).

Compound libraries are available from a number of commercial vendorssuch as Tocris BioScience, Nanosyn, BioFocus, and from governmententities. For example, the Molecular Libraries Small Molecule Repository(MLSMR), a component of the U.S. National Institutes of Health (NIH)Molecular Libraries Program is designed to identify, acquire, maintain,and distribute a collection of >300,000 chemically diverse compoundswith known and unknown biological activities for use, e.g., inhigh-throughput screening (HTS) assays (see https://mli.nih.gov/mli/).The NIH Clinical Collection (NCC) is a plated array of approximately 450small molecules that have a history of use in human clinical trials.These compounds are highly drug-like with known safety profiles. The NCCcollection is arrayed in six 96-well plates. 50 μl of each compound issupplied, as an approximately 10 mM solution in 100% DMSO. In someembodiments, a collection of compounds comprising “approved human drugs”is tested. An “approved human drug” is a compound that has been approvedfor use in treating humans by a government regulatory agency such as theUS Food and Drug Administration, European Medicines Evaluation Agency,or a similar agency responsible for evaluating at least the safety oftherapeutic agents prior to allowing them to be marketed. The testcompound may be, e.g., an antineoplastic, antibacterial, antiviral,antifungal, antiprotozoal, antiparasitic, antidepressant, antipsychotic,anesthetic, antianginal, antihypertensive, antiarrhythmic,antiinflammatory, analgesic, antithrombotic, antiemetic,immunomodulator, antidiabetic, lipid- or cholesterol-lowering (e.g.,statin), anticonvulsant, anticoagulant, antianxiety, hypnotic(sleep-inducing), hormonal, or anti-hormonal drug, etc. In someembodiments, a compound is one that has undergone at least somepreclinical or clinical development or has been determined or predictedto have “drug-like” properties. For example, the test compound may havecompleted a Phase I trial or at least a preclinical study in non-humananimals and shown evidence of safety and tolerability. In someembodiments, a test compound is substantially non-toxic to cells of anorganism to which the compound may be administered or cells in which thecompound may be tested, at the concentration to be used or, in someembodiments, at concentrations up to 10-fold, 100-fold, or 1,000-foldhigher than the concentration to be used. For example, there may be nostatistically significant effect on cell viability and/or proliferation,or the reduction in viability or proliferation can be no more than 1%,5%, or 10% in various embodiments. Cytotoxicity and/or effect on cellproliferation can be assessed using any of a variety of assays (some ofwhich are mentioned above). In some embodiments, a test compound is nota compound that is found in a cell culture medium known or used in theart, e.g., culture medium suitable for culturing vertebrate, e.g.,mammalian cells or, if the test compound is a compound that is found ina cell culture medium known or used in the art, the test compound isused at a different, e.g., higher, concentration when used in a methodof the present invention.

In some embodiments, a test compound is a compound that is recognized inthe art as having antiviral activity against one or more viruses, butthat is not known to be useful to inhibit infection by a virus ofinterest, e.g., a picornavirus. In some embodiments, a test compound isnot a compound that is recognized in the art as having antiviralactivity.

In some embodiments, one or more compounds or mixtures thereof havingknown antiviral activity is tested, wherein the molecular target of thecompound or mixture and/or mechanism of antiviral activity is unknown.Testing of such compounds or mixtures according to the present inventionto determine whether they inhibit PLA2G16 may lead to discovering thatPLA2G16 is the molecular target. Such discovery may facilitatepurification of an active component from a mixture, development of morehighly active derivatives of the compound, and/or otherwise permitfurther development of the compound or mixture as a therapeutic agent.

The step of determining whether a test compound inhibits PLA2G16expression can be carried out in a variety ways. Compounds that inhibitPLA2G16 expression can be identified by contacting cells with a testcompound, maintaining the cells in culture for a suitable period of time(e.g., sufficient time to allow degradation of existing PLA2G16 mRNAand/or protein), and then measuring the level of PLA2G16 mRNA orprotein. Methods known in the art can be used for measuring mRNA orprotein. A variety of different hybridization-based oramplification-based methods are available to measure RNA. Examplesinclude Northern blots, microarray (e.g., oligonucleotide or cDNAmicroarray), reverse transcription (RT)-PCR (e.g., quantitative RT-PCR),or reverse transcription followed by sequencing. The TaqMan® assay andthe SYBR® Green PCR assay are commonly used real-time PCR techniques.Other assays include the Standardized (Sta) RT-PCR™ (Gene Express, Inc.,Toledo, Ohio) and QuantiGene® (Panomics, Inc., Fremont, Calif.). In someembodiments the level of PLA2G16 mRNA is measured. In other embodiments,a reporter-based system is used. In some embodiments, a reporter-basedsystem comprises a nucleic acid in which expression control elements ofthe PLA2G16 gene are operably linked to a sequence that encodes areporter molecule (“reporter”). Reporters are often proteins but couldbe nucleic acids. Reporters are often readily detectable molecules, suchas proteins that produce a fluorescent, luminescent, or colorimetricsignal or are capable of absorbing light of a particular wavelength. Insome embodiments, a reporter molecule comprises an enzyme that acts on asubstrate to produce a fluorescent, luminescent, or colorimetric signal.Exemplary reporter molecules include, e.g., green, blue, sapphire,yellow, red, orange, and cyan fluorescent proteins and derivativesthereof; monomeric red fluorescent protein and derivatives such as thoseknown as “mFruits”, e.g., mCherry, mStrawberry, mTomato; luciferase;beta-galactosidase; horseradish peroxidase; alkaline phosphatase; etc.In some embodiments, a reporter is a secreted protein. In someembodiments, a reporter is encoded by a sequence that is codon-optimizedfor expression in a cell from an organism of interest. Methods forassessing the efficacy of an RNAi agent to silence expression of atarget gene can involve use of a sequence in which the mRNA target of anshRNA or siRNA (or a portion of the target) is cloned downstream of asequence that encodes a reporter, so that a bicistronic mRNA transcriptencoding both the target sequence and the reporter is produced. Targetgene knockdown results in the degradation (or translational inhibition)of the mRNA transcript, which causes a proportional decrease in theexpression of the reporter protein.

Compounds that inhibit PLAG16 molecular function can be identified usinga variety of different cell-free or cell-based assays. A cell-free assaytypically involves an isolated target molecule. For example, the targetmolecule could be present in a cell or tissue lysate or fraction thereof(e.g., a lysate made from cells that express the target molecule) orcould be an at least partially purified or synthesized target molecule.A tissue lysate could be made from any tissue containing cells thatexpress PLA2G16. In some embodiments, a tissue lysate is obtained fromadipose tissue, e.g., white adipose tissue. Various cells from which acell lysate could be prepared or from which a PLA2G16 polypeptide couldbe purified are mentioned below in the discussion of cell-based assays.In some embodiments, an isolated polypeptide is a polypeptide that hasbeen synthesized using recombinant nucleic acid techniques or in vitrotranslation. In order to perform the assay, a test compound is contactedwith the target molecule, e.g., by preparing a composition comprisingthe test compound and the target molecule. One or more parameters aremeasured, e.g., binding, enzymatic activity, etc. The composition cancomprise other component(s) necessary or helpful for identifying acompound of interest. In some embodiments, a composition for use in abinding assay or activity assay comprises cell membranes or cellmembrane components. Such membranes or components may be naturallyoccurring (e.g., components present in animal cell membranes),articifical, or combination thereof in various embodiments. For example,the composition can contain a lipid membrane bilayer, lipid vesicles,etc. Optionally, a lipid bilayer is immobilized on a surface. In someembodiments the lipids comprise phospholipids.

A variety of cell-free assays may be performed to identify compoundsthat inhibit a PLA2G16 polypeptide. In some embodiments, an assaydetects whether a test compound binds to a PLA2G16 polypeptide and/orquantifies one or more characteristics of such binding. Numerous bindingassay formats are known in the art. In some embodiments, a label-freeassay is used, while in other embodiments either the PLA2G16 polypeptideor test compound is detectably labeled. In some embodiments, a PLA2G16polypeptide or a compound to be tested for ability to bind to and/orinhibit activity of a PLA2G16 polypeptide is attached to a solidsupport. In some embodiments, a solid support is an article having arigid or semi-rigid surface. In some embodiments, at least one surfaceof the support is substantially flat. In other embodiments, a support isapproximately spherical. A support can be composed of an inorganic ororganic material or combination thereof. In some embodiments, a supportis composed at least in part of a metal, ceramic, glass, plastic, gel,or other matrix. Such articles may, for example, take the form of plates(e.g., multiwell plates), slides, particles (e.g., “beads”, e.g.,magnetic beads), pellets, bars, rods, pins, disks, chips, filters, orother suitable forms. In some embodiments, a support comprises a sensor,e.g., a sensor capable of detecting changes in binding. For example, thesensor could detect a change in weight or a signal such as fluorescence.In some embodiments, the support comprises an electrode. In someembodiments, compounds are arranged as a small molecule microarray.Compounds could be present in multiple locations on a surface, inindividual wells or vessels, etc. See, e.g., Vegas A J, et al., Chem SocRev. 37(7):1385-94, 2008. In some embodiments, a PLA2G16 polypeptide orcompound is noncovalently attachment or covalently linked to thesupport. Noncovalent attachment could be, e.g., by adsorption of thepolypeptide or compound to the surface (which may be coated with asubstance to facilitate such adsorption), via an affinity-basedmechanism, or other means of immobilizing the PLA2G16 polypeptide ortest compound so that it remains physically associated with the support.In some embodiments, an antibody is used to attach a PLA2G16 polypeptideor test compound to a support. In some embodiments, a PLA2G16polypeptide or test compound is attached to a support via abiotin-avidin interaction or other strong binding interaction, whereinone of two binding partners is attached directly or indirectly to thesupport and the other binding partner is attached to the molecule to beimmobilized.

In some embodiments, test compounds are immobilized in multiplelocations (e.g., in an array format. PLA2G16 polypeptide is added andthe composition is maintained for a suitable time period to allowbinding to occur. In some embodiments, unbound material is removed bywashing, and PLA2G16 polypeptide is detected using an antibody or, ifthe polypeptide is detectably labeled, by detecting a signal. In otherembodiments, a washing step is not necessary. For example, binding maybe detected by measuring a change in fluorescence polarization,fluorescence resonance energy transfer, or electrochemiluminescence. Inother embodiments, PLA2G16 polypeptide is immobilized, test compoundsare added, and binding is measured using similar approaches.

In some embodiments, surface plasmon resonance (SPR) is used to measurekinetics (on and/or off rates) and/or binding strength (affinity)between a test compound and a PLA2G16 polypeptide. For example, usingSPR technology (e.g., systems such as those available from Biacore, LifeSciences, GE Healthcare) the binding and dissociation of a test compoundto a protein immobilized on a chip can be measured, and the measuredvalues compared with those obtained when a solution not containing thetest compound is loaded on the chip. A test compound capable of bindingto the protein can be selected on the basis of the binding anddissociation rate and/or binding level. Other useful methods fordetecting and/or quantifying binding include use of a quartz crystalmicrobalance, optical cantilever, microchannel resonator, dualpolarisation interferometer, coupled waveguide plasmon resonance,immunoprecipitation or other antibody-based detection methods,isothermal titration and differential scanning calorimetry, capillaryelectrophoresis, resonance energy transfer, electrochemiluninesce, andfluorescent correlation analysis.

In some embodiments, an aptamer, peptide, non-hydrolyzable substrateanalog, or small molecule that is known to bind to a PLA2G16 polypeptideis labeled and used as a tool for screening test compounds (e.g., smallmolecules) for ability to bind to and/or inhibit activity of thepolypeptide. The label can comprise, e.g., a radioactive, fluorescent,luminescent, or other readily detectable moiety. The ability of a testcompound to compete with the labeled aptamer, peptide, non-hydrolyzablesubstrate analog, or small molecule can be detected and serves as anindicator of the binding of the test compound to the PLA2G16polypeptide. For example, a scintillation proximity assay (SPA) can beused. In some embodiments of an SPA for identifying compounds that bindto a PLA2G16 polypeptide, the PLA2G16 polypeptide is attached to beadscontaining a scintillant material. The beads are typically located inwells or other vessels. In another embodiment, a PLA2G16 polypeptide isattached to scintillant material is embedded directly into wells. Aradiolabelled compound capable of binding to the PLA2G16 polypeptide anda test compound are added to the well. Binding of the radiolabelledcompound to the PLA2G16 polypeptide results in a signal. The signal isreduced in the presence of a test compound that competes with theradiolabelled compound for binding. See, e.g., J. Fraser Glickman, etal., Scintillation Proximity Assays in High-Throughput Screening. Assayand Drug Development Technologies. 6(3): 433-455, 2008, for review ofSPA. Similar assays can be performed using filters.

In some embodiments, a compound is selected that binds to PLA2G16polypeptide with a Kd equal to or less than approximately 1 mM, 500 μM,100 μM, 50 μM, 10 μM, 5 μM, or 1 μM. In some embodiments, a compoundbinds to a PLA2G16 polypeptide with a Kd equal to or less thanapproximately 500 nM, 100 nM, 50 nM, or 10 nM. In some embodiments, acompound binds to a PLA2G16 polypeptide with a Kd between 0.1-10 nM.Compounds that bind to a PLA2G16 polypeptide may be further tested,e.g., in cell-free or cell-based assays, to determine the extent towhich they inhibit PLA2G16 activity (e.g., catalytic activity), e.g., asdescribed below.

A variety of different assays can be employed to identify and/orcharacterize compounds that inhibit PLA2G16 activity. In someembodiments, the ability of a compound to inhibit catalysis of achemical reaction by PLA2G16 polypeptide is assessed. In someembodiments, the chemical reaction is hydrolysis of the sn-2 bond of aphospholipid. A composition comprising a PLA2G16 polypeptide, one ormore PLA2G16 substrate(s), and a test compound is provided. The PLA2G16polypeptide, one or more PLA2G16 substrate(s), and test compound areusually in a suitable liquid medium. In some embodiments, the liquidmedium is an aqueous medium that comprises at least 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99% or more water (v/v). In some embodiments,the liquid medium may comprise an organic solvent such as DMSO, e.g., inan amount that does not significantly affect the activity of the PLA2G16polypeptide as compared with the activity in the absence of the organicsolvent. A “substrate” in this context is a molecule on which PLA2G16acts, i.e., a molecule that undergoes a chemical reaction that iscatalyzed by PLA2G16. Exemplary substrates are discussed below. Theconcentration of the substrate and PLA2G16 polypeptide can vary. In someembodiments, the substrate is present at between about 1 μM and 500 μM,e.g., between about 10 μM and about 50 μM, 100 μM, or 200 μM. In someembodiments, the PLA2G16 polypeptide is present at between 1 μg/ml andabout 100 μg/ml. It will be understood that the selection ofconcentrations and amounts can depend at least in part on the particularassay and is within the skill in the art. The composition is maintainedfor a suitable time period under conditions that would otherwise (i.e.,in the absence of a compound that is a potential PLA2G16 inhibitor) beappropriate for the PLA2G16 polypeptide to catalyze a reaction in whichthe substrate(s) is/are converted to one or more product(s). Thereaction may be stopped after a desired time period, e.g., by additionof (2:1) methanol:chloroform. The conditions and other component(s)present in the composition can vary depending, e.g., on the particularassay. Suitable conditions for a PLA2G16 polypeptide to act on asubstrate can include, e.g., a pH of between about 6.5 and about 9.5,e.g., between about 7.0 and about 9.0, e.g., between about 7.5 and about8.5, e.g., about 8.0. In some embodiments, the temperature is between10° C. and 40° C., e.g., between 20° C. and 30° C., e.g., about 25° C.Other components may be present in the composition. In some embodiments,the composition comprises a buffer substance such as Tris-HCl or sodiumborate, to help regulate the pH. Other buffer substances include, e.g.,HEPES, MOPS, etc. In some embodiments, the composition comprises adivalent cation, e.g., calcium (Ca2+). For example, in some embodiments,the composition comprises up to about 5 mM calcium, e.g., between about0.5 mM and about 2.5 mM calcium. In exemplary embodiments, thecomposition comprises about 1 mM calcium or about 2 mM calcium. In someembodiments, the composition does not comprise a calcium chelator suchas EDTA. In some embodiments, the composition comprises a calciumchelator in an amount that does not reduce the free calciumconcentration below about 1.0 mM. In some embodiments, the compositioncomprises a detergent, e.g., deoxycholate, e.g., at between 1-5 mM,e.g., about 2 mM or about 3 mM.

In some embodiments, the amount of product produced and/or the rate ofproduct formation is determined. The effect of the test compound on theamount of product produced and/or the rate at which the product isproduced is assessed, e.g., by comparison with a suitable referencevalue. If the amount of product or rate of product production isdecreased in the presence of the test compound as compared with asuitable reference value, the test compound inhibits the ability of thePLA2G16 polypeptide to catalyze a reaction in which the substrate isconverted to one or more product(s), i.e., the test compound is aninhibitor of the PLA2G16 polypeptide. In some embodiments, the rate ofsubstrate consumption or the amount of substrate consumed is determined.Equivalently, the amount of substrate remaining can be determined. Ifthe amount of substrate consumed or the rate of substrate consumption isdecreased in the presence of the test compound as compared with asuitable reference value, the test compound inhibits the ability of thePLA2G16 polypeptide to catalyze a reaction in which the substrate isconverted to one or more product(s), i.e., the test compound is aninhibitor of the PLA2G16 polypeptide. A reference value in any of theseassays can be a value measured under similar or identical conditions inthe absence of the test compound.

In some embodiments, a PLA2G16 substrate is useful to measurephospholipase activity, e.g., phospholipase A2 activity. For example, aPLA2G16 substrate can be a naturally occurring or artificialphospholipid. As known in the art, most phospholipids are composed of1,2-diacylglycerol and a phosphate group, and an organic molecule (oftena nitrogenous base). A phosophodiester bridge links the glycerolbackbone to the base, which is sometimes termed a “head group”.Exemplary head groups are choline, ethanolamine, inositol, and serine.For example, a substrate can be a phosphatidylcholine orphosphatidylethanolamine. The hydrocarbon chains of the acyl groups of aphospholipid molecule are often different, e.g., they are derived fromfatty acid molecules with different hydrocarbon chains. In someembodiments, the hydrocarbon chains are between 12 and 30 carbons inlength. In some embodiments, a PLA2G16 substrate has the structure of anaturally occurring phospholipid, e.g., a phospholipid found invertebrate cells, e.g., mammalian cells. Exemplary PLA2G16 substratesinclude, e.g., 1-palmitoyl-2-linoleoyl-PC, dilinoleoyl-PC,1-palmitoyl-2-linoleoyl-PS, 1-palmitoyl-2-linoleoyl-PE,phosphatidylinositol, 1-palmitoyl-2-arachidonyl-PC (abbreviations: PC:phosphatidylcholine PE: phosphatidylethanolamine; PS:phosphatidylserine). In some embodiments, the substrate comprisescholine as a head group. In some embodiments, a phospholipid analogcontaining a thio ester bond instead of the sn-2 ester is used. In someembodiments, a PLA2G16 substrate is useful for measuringlysophospholipase activity. For example, the substrate can be alysophosphatidylcholine, e.g.,1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine.

In some embodiments, a substrate comprises a moiety that facilitatesdetection of a product of a biochemical reaction catalyzed by a PLA2G16polypeptide. For example, the substrate can comprise one or moreradioactive atoms, fluorescent labels, and/or fluorescence quenchers. Insome embodiments, the label comprises 14C, 3H, or 32P. In someembodiments, the substrate comprises a moiety that emits a signal uponcleavage of the substrate. In some embodiments, the substrate comprisesa moiety that can be readily detected upon release from the substrate.For example, the moiety may react with another compound to produce acolorimetric, fluorescent, or luminescent signal. Labels include, e.g.,organic materials (including “traditional” dye fluorophores, quenchers,and polymers); inorganic materials such as metal chelates, metal andsemiconductor nanocrystals (e.g., “quantum dots”, and fluorophores ofbiological origin such as certain amino acids (e.g., tryptophan,tyrosine); and compounds that exhibit luminescensce upon enzymaticcatalysis such as naturally occurring or synthetic luciferins (e.g.,firefly or Renilla luciferin, coelenterazine). Fluorescent dyes include,e.g., acridine dyes; Alexa dyes; BODIPY, cyanine dyes; fluorescein dyes,rhodamine dyes, and derivatives of any of the foregoing. See, e.g., TheHandbook—A Guide to Fluorescent Probes and Labeling Technologies, 10thedition (Invitrogen Corp.), which describes numerous fluorescent andotherwise detectable molecules and methods for their use andmodification. In another embodiment, a phospholipid analogue containinga thio ester bond instead of the sn-2 ester is used, and hydrolysis ofthe thioester bond at the sn-2 position by PLA2 releases free thiolwhich can be detected by DTNB (5,5′-dithiobis(2-nitrobenzoic acid)).

In some embodiments, a substrate is present in a vesicle or micelle. Forexample, lipid-detergent micelles can be used. In some embodiments, anionic detergent such as deoxycholate is used. Other detergents include,e.g., Triton X-100. In some embodiments, a composition containing about100 μM 1-palmitoyl-2-linoleoyl-PC with 2 mM deoxycholate and 2 mM CaCl₂is used. A test compound can be incorporated into the vesicle ormicelle.

A variety of assays can be used to measure PLA2 catalytic activity. Insome embodiments, an assays that has been used in the art to measureactivity of a Group I-XV PLA2 (e.g., a cytosolic or secreted PLA2) isused or modified for use to measure catalytic activity of PLA2G16. Insome embodiments, a radiometric assay is used, with a substrate ofphospholipid (e.g., phosphatidylcholine or phosphatidylethanolamine)containing a 14C- or 3H-labeled fatty acid at the sn-2 position. Thefatty acids released are separated from the unreacted substrate andquantified by liquid scintillation counting. In other embodiments, afluorescence displacement assay, is used. A fluorescent molecule can bedetected using, e.g., a spectrophotometer. An exemplary assay involvesthe displacement of a fluorescent fatty acid probe from albumin or ratliver fatty acid-binding protein by the decanoic acid released as aresult of the phospholipase A2-catalyzed hydrolysis ofdidecanoyl-phosphatidylcholine A. R. Kinkaid & D. C. Wilton, Acontinuous fluorescence displacement assay for phospholipase a2 usingalbumin and medium chain phospholipid substrates. Anal. Biochem. 212:65-70, 1993; D. C. Wilton, A continuous fluorescence displacement assayfor the measurement of phospholipase A2 and other lipases that releaselong-chain fatty acids. Biochem. J. 266: 435-439, 1990). See also,Huang, Z., et al., Anal. Biochem. 222: 110-115, 1994, which describes anassay for cPLA2 activity based on hydrolysis of fatty acid esters of7-hydroxycoumarin by cPLA2, producing the free fatty acid and highlyfluorescent 7-hydroxycoumarin. Another assay is a fluorometricphospholipase assay based on polymerized liposome substrates (Chu, W.,et al., Fluorometric phospholipase assays based on polymerised liposomesubstrates. Methods Mol. Biol. 109: 7-17, 1999). In another embodiment,a phospholipid analogue containing a thio ester bond instead of the sn-2ester is used as a substrate to measure phospholipase activity (Yu, L,et al. Carbonothioate phospholipids as substrate for aspectrophotometric assay of phospholipase A2. Anal. Biochem. 265: 35-41,1998).

In another embodiment, a coupled spectrophotometric assay usingdilinoleoyl phosphatidylcholine (DL-PC) as PLA2 substrate andlipoxygenase as the coupling enzyme is used. See, e.g., Jiménez, M., etal. A continuous spectrophotometric assay for phospholipase A(2)activity Anal Biochem., 319(1):131-7, 2003, and references therein, andDuncan, supra. In this assay, lipoxygenase (linoleate:oxygenoxidoreductase, EC 1.13.11.12) catalyzes the addition of molecularoxygen to fatty acids containing at least one (Z,Z)-pentadiene system togive the corresponding hydroperoxides. Lipoxygenase oxidizes thelinoleic acid released by the action of phospholipase, the activity ofwhich can then be followed spectrophotometrically by recording theincrease in absorbance at 234 nm due to the formation of thecorresponding hydroperoxide from the linoleic acid by the action oflipoxygenase. This method provides a continuous record of phospholipidhydrolysis.

In some embodiments, a scintillation proximity assay (SPA) is used. Forexample, a radiolabelled PLA2G16 substrate can be attached to beadscontaining a scintillant material. The beads are typically located inwells or other vessels. In another embodiment, scintillant material isembedded directly into wells. A PLA2G16 polypeptide is added to the wellin a suitable composition (optionally containing calcium and/or abuffer). Hydrolysis of the substrate releases the radioactive moiety,resulting in a decreased signal. See, e.g., J. Fraser Glickman, suprafor discussion of SPA.

In some embodiments, an assay readout is based on resonance energytransfer (RET), e.g., fluorescence resonance energy transfer (FRET),luminescence resonance energy transfer (LRET), or bioluminescenceresonance energy transfer (BRET). A wide variety of RET-based assays canbe implemented. In general, such assays make use of a distance-dependentinteraction involving energy transfer between two moieties (sometimestermed a donor and acceptor). If both moieties are present as part of aPLA2G16 substrate and positioned so that cleavage of the substratereleases one of the moieties, a signal (e.g., an increase or decrease ina signal) can be detected. FRET is a distance-dependent interactionbetween the electronic excited states of two moieties in whichexcitation is transferred from a donor moiety to an acceptor moietywithout emission of a photon, resulting in emission from the FRETacceptor. LRET has similarities to FRET but uses a luminescent moiety,e.g., a lanthanide as the energy-transfer donor. BRET is analogous toFRET but uses a luminescent or luminescence-generating biomolecule suchas luciferase, aequorin, or a derivative thereof as an energy donor anda fluorescent moiety, e.g., a biomolecule such as green fluorescentprotein (GFP) as the acceptor, thus eliminating the need for anexcitation light source (reviewed in Pfleger, K. an Eidne, K., NatureMethods, 3(3), 165-174, 2006).

Assays of the invention may detect acceptor emission, donor quenching(decreased emission from the RET donor), and/or an alteration in thefluorescence lifetime of the donor. Assays of the invention can make useof increases in acceptor emission, decreases in acceptor emission, donorquenching, reduction in donor quenching, and/or increase or decrease influorescence lifetime of the donor to detect cleavage of a PLA2G16substrate. Nonfluorescent acceptors, also referred to as quenchers areof use and include dabcyl and QSY dyes. Such molecules are capable ofabsorbing the energy of an excited fluorescent label when located inclose proximity and of dissipating that energy without the emission ofvisible light. Numerous suitable donor/acceptor pairs are known in theart. See, e.g, The Handbook—A Guide to Fluorescent Probes and LabelingTechnologies, 10th edition (Invitrogen Corp.).

In some embodiments of a FRET-based assay, a first acyl chain of thePLA2G16 substrate has an attached fluorescence quencher and the secondacyl chain has an attached fluorophore. Intramolecular FRET from thefluorophore to the quencher quenches fluorescence until PLA2-mediatedsubstrate cleavage, when at least one fatty acid moiety becomesseparated from the remainder of the molecule, and the intermoleculardistance exceeds that required for efficient energy transfer. Anincrease in fluorescence signal indicates substrate cleavage. Thepresence of an inhibitor will cause a reduction in the fluorescencesignal relative to that which would be observed in the absence of theinhibitor. In some embodiments the phospholipids sn-1-acyl chaincontains an attached fluorescence quencher (e.g., Dabcyl, also known asp-methyl red), and the sn-2 acyl chain contains an appended BODIPYfluorophore. Intramolecular FRET (fluorescence resonance energytransfer) to the Dabcyl group quenches BODIPY fluorescence untilPLA-mediated substrate cleavage. See, e.g., Rose, T M & Prestwich, G D,ACS Chemical Biology, 1(2): 83-89, 2006, for description of Dabcyl- andBODIPY-containing phospholipids DBPA, DBPC, DBPE, and DBPG(abbreviations: DB: Dabcyl-BODIPY; PG: phosphatidylglycerol).

Another assay format that can be used to measure PLA2 activity is afluorescence based assay in which cationic conjugated polyelectrolytesare supported on silica microspheres. (See, e.g., Chemburu S, et al.Conjugated polyelectrolyte supported bead based assays for phospholipaseA2 activity, Phys Chem B., 112(46):14492-9, 2008, which describes suchan assay for human serum-derived PLA2. This assay can be modified foruse to detect compounds that inhibit activity of a PLA2G16polypeptide.). The polymer-coated beads are overcoated with an anionicphospholipid to provide “lipobeads” that serve as a sensor for PLA2. Thelipid serves a dual role as a substrate for PLA2 and an agent toattenuate quenching of the polymer fluorescence by the external electrontransfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS).Quenching of the polymer fluorescence by AQS increases as the PLA2digests the lipid. The lipid can also be used itself as a quencher andsubstrate by employing a small amount of energy transfer quenchersubstituted lipid in the anionic phospholipid coating the beads. In thiscase the fluorescence of the polymer is quenched when the lipid layer isintact; as the enzyme digests the lipid, the fluorescence of the polymeris restored. The sensing of PLA2 activity can be performed by monitoringfluorescence changes in a multiwell plate reader and/or by flowcytometry.

A “cell-based assay” is an assay in which viable cells that express orcontain a PLA2G16 polypeptide are contacted with a test compound and aparameter of interest such as PLA2G16 activity is assessed. Typically,the cells are maintained in cell culture and the test compound is addedto the culture medium. In some embodiments, the effect of the testcompound on the ability of the PLA2G16 polypeptide to act on a PLA2G16substrate is assessed. For example, a PLA2G16 substrate, e.g, adetectably labeled substrate, can be added to the culture medium orsynthesized by the cell from a labeled precursor. Cleavage of thesubstrate may be detected by detecting a free fatty acid or by detectinga downstream product produced from a free fatty acid. For example,arachidonic acid is modified by cyclooxygenases to form eicosanoids(e.g., prostaglandins, leukotrienes). In some embodiments, a cell thatsubstantially lacks other PLA2 enzymes that could act on the PLA2G16substrate can be used. In some embodiments, such cells are identified byscreening a variety of cell lines for expression of known PLA2 enzymes.In other embodiments, a cell line is generated by targeted deletion orinsertion into the genes encoding one or more PLA2 enzyme(s) or bycausing the cell to express shRNA that inhibit expression of such otherPLA2 enzyme(s). In other embodiments, the assay is performed in cellsthat have been contacted with siRNA specific for such other PLA2enzyme(s) to knock down their expression.

A compound identified as an inhibitor of a PLA2G16 polypeptide can betested in cell culture or in animal models (“in vivo”) to determine itsability to inhibit viral infection. In some embodiments, host cells arecontacted with a virus and a PLA2G16 inhibitor under conditions suitablefor infection of the cells. The ability of the test compound to inhibitviral infection is assessed. If the compound detectably reduces viralinfection, the compound is identified as an antiviral compound. Thevirus can be, e.g., any virus that utilizes PLA2G16 polypeptide, or aPLA2G16-like polypeptide, in its life cycle.

A wide variety of cell types can be used in embodiments of the inventivemethods. Typically, the cell expresses or contains a PLA2G16polypeptide, either naturally or as a result of modification by the handof man, although cells that do not express a PLA2G16 may be useful,e.g., for control purposes. A cell could originate from any organism ofinterest, e.g., a vertebrate, e.g., a mammal. In some embodiments, acell is a primate cell, e.g., a monkey cell or a human cell. A cellcould be a primary cell, immortalized cell, cancer cell, etc. Often, acell is a member of a population of cells which is composed of cellsthat are substantially genetically identical, e.g., a cell line. A cellline can be descended from a single cell or from multiple cells isolatedfrom a single individual. A cell can originate from a tissue or organ ofinterest or can have a property of interest. In some embodiments, a cellis an epithelial cell, fibroblast, kidney cell, rhabdosarcoma orrhabdomyosarcoma, lung, or bronchial cell, pre-adipocyte, or adipocyte.In some embodiments a cell originates from breast, bladder, bone, brain,bronchus, cervix, colon, endometrium, esophagus, larynx, liver, lung,nerve, muscle, ovary, pancreas, prostate, stomach, kidney, skin, testis,or thyroid gland. Numerous cell lines are known in the art, many ofwhich can be obtained from repositories such as the American TypeCulture Collection, Coriell Cell Repositories, European Collection ofCell Cultures, Japanese Collection of Research Bioresources, or from avariety of commercial suppliers. In some embodiments, a pre-adipocyte isa 3T3-L1 cell. In some embodiment, a cell is a COS cell, e.g., a COS-1or COS-7 cell. In some embodiments, a cell is a HeLa cell. In someembodiments, a cell is a Vero, RD, CHO, HEK-293, HMEC, MDCK, NIH-3T3,HEp-2, A549, or BEAS-2B cell. In some embodiments, a cell is a tumorcell. In some embodiments a tumor cell originates from a carcinoma. Insome embodiments a tumor cell originates from a sarcoma. In someembodiments a tumor cell originates from a hematologic malignancy, e.g.,a lymphoma or leukemia or myeloma. In some embodiments a tumor celloriginates from a breast, bladder, bone, brain, cervical, colon,endometrial, esophageal, head and neck, laryngeal, liver, lung (smallcell or non-small cell), ovarian, pancreatic, prostate, stomach, renal,skin (e.g., basal cell, melanoma, squamous cell), testicular, or thyroidcancer. The tumor cell may be a cell of an established tumor cell line(e.g., one of the NCI-60 tumor cell lines) or another tumor cell lineknown in the art or newly established.

In some embodiments, a cell is a hematopoietic cell. In someembodiments, a cell is a KBM-7 cell or derivative thereof, such as aHAP1 cell. In some embodiments, a cell is a KBM-7 cell or other cellthat has been partially reprogrammed by expressing at least one“reprogramming factor” therein or exposing the cell to at least one“reprogramming agent” (e.g., an agent that induces expression of anendogenous reprogramming factor or substitutes for a reprogrammingfactor). Reprogramming cells, e.g., near-haploid mammalian cells mayfacilitate their use in identifying PLA2G16 inhibitors and/or antiviralcompounds. Such reprogramming may convert the KBM-7 cell (which normallygrows in suspension) into an adherent cell, such as a HAP1 cell. Asknown in the art, mouse and human fibroblasts and various other normalsomatic cell types can be reprogrammed in vitro to a pluripotent statethrough retroviral-mediated introduction of combinations oftranscription factors, e.g., the four transcription factors Oct4, Sox2,Klf4, and c-Myc (with c-Myc being dispensable, although omitting c-Mycreduced reprogramming efficiency), or the four transcription factorsOct4, Nanog, Sox2, and Lin28 (see, e.g., Meissner, A., et al., NatBiotechnol., 25(10):1177-81 (2007); Yu, J., et al, Science,318(5858):1917-20 (2007); and Nakagawa, M., et al., Nat Biotechnol.,26(1):101-6 (2008). Such transcription factors are often referred to as“reprogramming factors”).

In some embodiments, a cell naturally expresses PLA2G16. In someembodiments a cell is modified so that it expresses a PLA2G16polypeptide at a higher level than would be the case in the absence ofthe modification. In some embodiments, a cell expresses PLA2G16 at alevel at least 25%, 50%, 75%, 90%, 95%, or approximately 100% as high asthe expression level present in a HAP1 cell, HeLa cell, or other cellcapable of serving as a host cell for a virus of interest. Theexpression level can be normalized, e.g., based on expression of a“housekeeping” gene. Commonly used housekeeping genes include, e.g.,beta-actin, GAPDH, phosphoglycerate kinase, etc. Standard methods oftransiently or stably expressing polypeptides in cells can be used.

In some embodiments, a cell is of a type that is known in the art to benaturally susceptible to infection by a virus, e.g., a picornavirus. Forexample, the cell can be of a type that is normally a target cell of thevirus in vivo or a cell line that has been used in the art as a host fora virus in culture. A compound can be tested in cells of multipledifferent types. For example, a compound can be initially identified asa PLA2G16 inhibitor or antiviral compound in a cell that has convenientproperties for screening or performing tests for virus inhibition andthen subsequently tested in one or more cells that are natural targetsof a virus of interest.

In some embodiments, a cell used in a method described herein isgenetically modified or selected to have a property that facilitates itsuse to test compounds. For example, the cell can be genetically modifiedor selected to have reduced or absent expression of one or moremolecular pumps that may otherwise transport a test compound out of thecell. In some embodiments, the cell is modified to facilitate detectionof viral infection. For example, the cell could comprise a reporter genein which a promoter or other expression control element(s) active onlyin the presence of viral protein(s) are operably linked to an openreading frame that encodes a readily detectable polypeptide such as afluorescent protein or enzyme. In another embodiment, a cell expresses aprotein that comprises a cleavage site for a viral protease, whereincleavage of the protein is detectable. For example, the protein maycontain a FRET pair (e.g., polypeptides that are a FRET donor andacceptor pair) separated by a domain containing a protease cleavagesite. Cleavage by the protease results in separation of the members ofthe FRET pair, resulting in a disruption of FRET, which can be detectedand serve as an indicator of viral infection. In another embodiment, acell-permeable substrate for a viral protease is introduced into thecells. A candidate antiviral compound, e.g., a compound that inhibitsPLA2G16 activity, can be tested in such cells to confirm that itinhibits viral infection.

Cells can be contacted with test compound(s) and/or virus for variousperiods of time. In certain embodiments cells are contacted with testcompound(s) and/or virus for between 1 hour and 20 days, e.g., forbetween 12 and 48 hours, between 48 hours and 5 days, e.g., about 3days, between 5 days and 10 days, or any intervening range or particularvalue. In some embodiments, cells are contacted with a virus for atleast a time sufficient for completion of one or more rounds of viralreplication and production of progeny virus. In some embodiments, cellsare contacted with a virus for at least a time sufficient for productionof plaques that are detectable under a light microscope. Cells can becontacted with a test compound during all or part of a culture period.If desired, the test compound can be removed prior to assessing PLA2G16activity or viral infection. In some embodiments, cells are contactedwith a virus prior to contacting the cells with the test compound. Inother embodiments, cells are contacted with the test compound prior tocontacting them with a virus. The absolute number of virus and themultiplicity of infection (MOI) can vary. “Multiplicity of infection”refers to the ratio of infectious agents (e.g., viruses) to infectiontargets (e.g., cells). In some embodiments an MOI of between 10⁻⁴ and10² is used. For example, an MOI of between 0.001 and 10, e.g., between0.01 and 1, can be used. In some embodiments, an amount of virussuitable to produce a pathologic change in between 10% and 100% of cellsis used. One of skill in the art will be able to determine a suitableamount of virus to use so as to be able to detect an effect on viralinfection. A range of dilutions of a virus stock can be tested toidentify an appropriate amount. Cells are maintained in culture for asuitable time period after contacting them with the virus. Typically,the time period will be sufficient for the virus to enter cells and forat least one event indicative of viral infection to occur. Such eventmay be a detectable effect of a viral gene product(s) on the cell and/orthe synthesis or partial synthesis of at least one viral gene product.In general, the time period will be sufficient to detect a differencebetween the effect of the virus on the cells in the absence of a PLA2G16inhibitor versus in the presence of a PLA2G16 inhibitor. A detectableeffect of a virus on a cell could be an alteration (e.g., a decrease) insynthesis of some or most cellular RNA(s) or protein(s), induction of anantiviral response (e.g., induction of interferon target gene(s) such asthe gene encoding 2′5′-oligoadenylate synthetase), a morphologicaleffect such as chromatin condensation, nuclear blebbing, proliferationof membranous vesicles; leakage of intracellular contents; cytotoxicity;cleavage of a substrate by a virus-specific enzyme (e.g., a protease),etc. Cytotoxicity can be assessed e.g., by detecting cell lysis (whichmay be evident as clear areas or “plaques” in a cell monolayer) or usingany of a variety of assays for cell viability and/or proliferation suchas a cell membrane integrity assay, a cellular ATP-based viabilityassay, a mitochondrial reductase activity assay, a BrdU, EdU, orH3-Thymidine incorporation assay, a DNA content assay using a nucleicacid dye, such as Hoechst Dye, DAPI, Actinomycin D, 7-aminoactinomycin Dor propidium iodide, a cellular metabolism assay such as AlamarBlue,MTT, XTT, and CellTitre Glo, etc. Plaque assays are a well establishedmeans of assessing viral titer and detecting the effect of compounds onviral infectivity. In some embodiments, a plaque assay involvesinoculating a standard viral stock into multiple identical cellcultures, e.g., grown in wells of a multiwell plate. A solidifyingagent, e.g., agarose, may be added to minimize spread of the virusthrough the culture medium. The viral titer of the stock is usuallypredetermined and is selected to yield a countable number of plaques ineach well. Different concentrations of the test compound are introducedinto a series of wells. The effect of the compound may be expressed asthe 50% inhibitory concentration (IC₅₀), defined as the lowestconcentration of compound that results in a 50% decrease in the numberof viral plaques compared with a control well that does not contain thecompound. If desired, an IC₉₀ can be assessed in a similar manner. Acompound that significantly decreases an effect of the virus is aninhibitor of infection by the virus. For example, a compound thatsignificantly decreases the number and/or size of viral plaques causedby a given amount of virus is an inhibitor of viral infection.Optionally, an IC50 or IC90 is determined. In some embodiments, one ormore compound(s) with a desired C50 or IC90 is selected. In someembodiments, an IC50 and/or IC90 is no greater than 100 mg/ml, e.g., nogreater than 10 mg/ml, e.g., no greater than 1.0 mg/ml, e.g., no greaterthan 100 μg/ml, e.g., no greater than 10 μg/ml, e.g., no greater than 5μg/ml or no greater than 1 μg/ml. In some embodiments, an IC50 and/orIC90 is less than or equal to 500 μM. In some embodiments, an IC50and/or IC90 less than or equal to 100 μM. In some embodiments, an IC50and/or IC90 less than or equal to 10 μM. In some embodiments, an IC50and/or IC90 is in the nanomolar range, i.e., less than or equal to 1 μM.

In some embodiments, a high throughput screen (HTS) is performed. A highthroughput screen can utilize cell-free or cell-based assays. Highthroughput screens often involve testing large numbers of compounds withhigh efficiency, e.g., in parallel. For example, tens or hundreds ofthousands of compounds can be routinely screened in short periods oftime, e.g, hours to days. Often such screening is performed in multiwellplates containing, e.g., e.g., 96, 384, 1536, 3456, or more wells(sometimes referred to as microwell or microtiter plates or dishes) orother vessels in which multiple physically separated cavities arepresent in a substrate. High throughput screens can involve use ofautomation, e.g., for liquid handling, imaging, data acquisition andprocessing, etc. Without limiting the invention in any way, certaingeneral principles and techniques that may be applied in embodiments ofa HTS of the present invention are described in Macarrón R & Hertzberg RP. Design and implementation of high-throughput screening assays.Methods Mol Biol., 565:1-32, 2009 and/or An W F & Tolliday N J.,Introduction: cell-based assays for high-throughput screening. MethodsMol Biol. 486:1-12, 2009, and/or references in either of these.Exemplary methods are also disclosed in High Throughput Screening:Methods and Protocols (Methods in Molecular Biology) by William P.Janzen (2002) and High-Throughput Screening in Drug Discovery (Methodsand Principles in Medicinal Chemistry) (2006) by Jorg H{umlaut over(υ)}ser.

In some embodiments, a first screen is performed to identify compoundsthat bind to and/or inhibit PLA2G16 polypeptide, and the ability of suchcompounds to inhibit viral infection is then assessed. In someembodiments, test compounds are first tested in a cell-based assay toidentify compound(s) that inhibit viral infection and are then tested todetermine whether they inhibit PLA2G16.

The invention provides compositions comprising components appropriate toperform any of the inventive methods, e.g., any of the methods ofidentifying a candidate antiviral compound. In some embodiments, anassay system comprises components suitable for identifying a PLA2G16inhibitor. In some embodiments, a composition comprises componentsappropriate to perform any of the inventive methods of validating acandidate antiviral compound. In some embodiments, the compositioncomprises components appropriate to confirm that a candidate antiviralcompound inhibits viral infection in cultured cells or in vivo. In oneaspect, an inventive composition comprises (i) isolated cells thatexpress a PLA2G16 polypeptide; (ii) a virus capable of infecting thecells; and (iii) a test compound. In some embodiments, the virus is aPicornavirus, e.g., a pathogenic Picornavirus. The virus is typicallypresent in the composition in amounts suitable for detecting virusinfection by the cells. Such amounts are typically greater than mighthappen by chance if cultured cells happen to be exposed to anenvironment where there is an individual infected by the virus. In someembodiments, the ratio of viral particles (e.g., infectious viralparticles) to cells is at least 1:10⁶, at least 1:10⁵, e.g., at least1:10⁴, at least 1:10³, at least 1:10², at least 1:10, or at least 1:1.In some embodiments, there are more viral particles (e.g., infectiousviral particles) than cells. The test compound can be, e.g, any of thecompounds discussed above. In some embodiments, the test compound is aphospholipase A2 inhibitor, e.g, a PLA2G16 inhibitor. In someembodiments, the test compound is a small molecule. In some embodiments,the test compound has been determined to bind to and/or inhibit PLA2G16in at least one cell-free or cell-based assay.

Compounds identified in cell-free and/or cell-based assays can be testedin subjects (e.g., non-human vertebrates) to assess their ability toinhibit viral infection in vivo. Animal models for viral infection areknown in the art. An animal can be, e.g., a rodent, non-human primate,dog, cat, etc. In one embodiment, an animal model is a murine model ofcoxsackievirus B3 (CVB3)-induced myocarditis. See, e.g., Szalay G,Ongoing coxsackievirus myocarditis is associated with increasedformation and activity of myocardial immunoproteasomes, Am J Pathol.,168(5):1542-52, 2006, and references therein. In one embodiment, ananimal model is a mouse model for EV71 infection. See, e.g. Wang, Y. F.,et al., A mouse-adapted enterovirus 71 strain causes neurologicaldisease in mice after oral infection. J. Virol. 78:7916-7924, 2004,which describe an animal model in which mice are orally inoculated withEV71. Mice may be monitored daily for signs of disease and survival. Inanother embodiment, an attenuated mengovirus is used in a rodent modelfor rhinovirus infection. See, e.g., Rosenthal L A, A rat model ofpicornavirus-induced airway infection and inflammation. Virol J., 6:122,2009. Tissues or body fluids can be collected after infection todetermine viral titers and/or to evaluate other signs of viralinfection. For example, viral RNA or protein can be detected usingstandard methods such as RT-PCR (for RNA) or immunological methods forproteins. See, e.g., Li, Z. H., et al., Ribavirin reduces mortality inenterovirus 71-infected mice by decreasing viral replication. J. Infect.Dis. 197:854-857, 2008).

The invention further provides a non-human subject e.g., a vertebrate,wherein the non-human subject has been inoculated with or exposed to avirus to which it is normally susceptible, or that is suffering from aviral infection, and wherein a PLA2G16 inhibitor has been administeredto the subject. “Inoculation” with a virus means that the virus has beenintroduced into the subject's body. Exposure can involve inoculating asubject or placing the virus and subject in reasonably close proximityso as to increase the likelihood that the subject will encounter thevirus. Inoculation can be by any appropriate route. Inoculation orexposure will typically involve sufficient amount of virus to produceevident disease in at least 25% of a population of that species in theabsence of an antiviral compound. The PLA2G16 inhibitor can be, e.g, anyof the compounds discussed above or identified according to an inventivemethod. In some embodiments, the test compound is a small molecule. Insome embodiments, the test compound has been determined to bind toand/or inhibit PLA2G16 in at least one cell-free or cell-based assay.The non-human subject can be monitored, e.g., to assess the safety,tolerability, and/or efficacy of the compound as an antiviral agent.Assessing the effect of a PLA2G16 inhibitor in a subject infected with avirus is an aspect of the invention.

In some embodiments, the invention provides a near-haploid cell that hasan insertion into the PLA2G16 locus or otherwise lacks expression ofPLA2G16. The near-haploid cell is of a species, e.g., a mammal, whosesomatic cells are normally diploid. In some embodiments, the inventionprovides a near-haploid cell that expresses a catalytically inactivemutant PLA2G16 polypeptide, wherein optionally the near-haploid mutantcell line has an insertion in the endogenous PLA2G16 gene. In someembodiments, the invention provides a near-haploid cell that expresses atagged functional PLA2G16 polypeptide, wherein optionally thenear-haploid mutant cell line has an insertion in the endogenous PLA2G16gene. A near-haploid mammalian cell, as used herein, refers to amammalian cell in which no more than 5 chromosomes are present in two ormore copies. In some embodiments a near-haploid mammalian cell has nomore than 1, 2, 3, or 4 chromosomes present in two or more copies. The“near-haploid” cell should be understood to include haploid cells.Further provided are cell lines derived from cells that lack expressionof functional PLA2G16, e.g., cell lines composed of cells that have aninsertion into the PLA2G16 gene. In some embodiments, a cell lineexpresses a catalytically inactive mutant PLA2G16 polypeptide, which istagged in some embodiments. In some embodiments, a near-haploid cellline eventually gains chromosomes during culture so that it is no longernear-haploid. In some embodiments the cell line may become near diploidor diploid.

The invention further provides kits comprising one or more components ofany of the inventive compositions and/or components suitable forperforming any of the inventive methods. The components can be packagedindividually, e.g., in individual containers, which may be providedwithin a larger container. A kit can contain instructions for using thecontents to perform any of the methods, e.g., to identify orcharacterize an antiviral compound.

In some embodiments, computational approaches are employed to identifyand/or characterize compounds that inhibit PLA2G16. For example, athree-dimensional structure of a PLA2G16 polypeptide can be determinedor an approximate structure generated using, e.g., nuclear magneticresonance, homology modeling, and/or X-ray crystallography. Optionallythe structure of the polypeptide with a ligand (e.g., an inhibitor)bound thereto is determined. In some embodiments, a computationalapproach is used in the initial identification of candidate PLA2G16inhibitors (sometimes termed “virtual screening”). Structures ofcandidate compounds can be screened for ability to bind to the PLA2G16polypeptide, e.g., to a region (e.g., a “pocket”) accessible to thecompound. The region could be a known or potential active site or anyregion accessible to the compound, e.g., a concave region on the surfaceor a cleft. A variety of docking and pharmacophore-based algorithms havebeen developed, and computer programs implementing such algorithms areavailable. Commonly used programs include Gold, Dock, Glide, FlexX,Fred, and LigandFit (including the most recent releases thereof). See,e.g., Ghosh, S., et al., Current Opinion in Chemical Biology, 10(3):194-2-2, 2006; McInnes C., Current Opinion in Chemical Biology; 11(5):494-502, 2007, and references in either of the foregoing articles, whichare incorporated herein by reference. In some embodiments, a virtualscreening algorithm involves two major phases: searching (also called“docking”) and scoring. During the first phase, the programautomatically generates a set of candidate complexes of two molecules(test compound and target molecule) and determines the energy ofinteraction of the candidate complexes. The scoring phase assigns scoresto the candidate complexes and selects a structure that displaysfavorable interactions based at least in part on the energy. To performvirtual screening, this process is repeated with a large number of testcompounds to identify those that display the most favorable interactionswith the target. In some embodiments, low-energy binding modes of asmall molecule within an active site or possible active site areidentified. Variations can include the use of rigid or flexible dockingalgorithms and/or including the potential binding of water molecules.

Numerous small molecule structures are available and can be used forvirtual screening. For example, ZINC is a publicly available databasecontaining structures of millions of commercially available compoundsthat can be used for virtual screening (http://zinc.docking.org/;Shoichet, J. Chem. Inf. Model., 45(1):177-82, 2005). A databasecontaining about 250,000 small molecule structures is available on theNational Cancer Institute (U.S.) website (athttp://129.43.27.140/ncidb2/). In some embodiments, multiple smallmolecules are screened, e.g., up to 50,000; 100,000; 250,000; 500,000,or up to 1 million, 2 million, 5 million, 10 million, or more. Compoundscan be scored and, optionally, ranked by their potential to bind to thetarget. Compounds identified in virtual screens can be tested incell-free or cell-based assays or in animal models to confirm theirability to inhibit PLA2G16 activity and/or viral infection.

Computational approaches can be used to predict one or morephysico-chemical, pharmacokinetic and/or pharmacodynamic properties ofcompounds identified in actual or virtual screens. For example,absorption, distribution, metabolism, and excretion (ADME) parameterscan be predicted. Such information can be used, e.g., to select hits forfurther testing or modification. For example, small molecules havingcharacteristics typical of “drug-like” molecules can be selected and/orsmall molecules having one or more undesired characteristics can beavoided. In one embodiment, compounds that satisfy at least some of theLipinski “rule of five” criteria are selected.

In one aspect, the invention provides a computer-readable medium onwhich are stored results of a screen to identify compounds that inhibitPLA2G16. The results may be stored in a database and can include anyscreening protocols, results obtained from the screen or from additionalscreens, and/or protocols of or results obtained from tests performed oncompounds identified in the screen (e.g., tests in animal models ofviral infection).

Additional compounds that inhibit PLA2G16 can be identified or designedbased on initial compounds (“hits”) identified in an actual or virtualscreen such as those described above. Such additional compounds andmethods of designing or synthesizing them are an aspect of theinvention. In some embodiments, structures of hit compounds are examinedto identify a pharmacophore, which can be used to design additionalcompounds (“derivatives”).

An additional compound may, for example, have one or more improved (i.e,more desirable) pharmacokinetic and/or pharmacodynamic properties ascompared with an initial hit or may simply have a different structure.For example, a compound may have higher affinity for the moleculartarget of interest (e.g., PLA2G16), lower affinity for a non-targetmolecule, greater solubility (e.g., increased aqueous solubility),increased stability, increased bioavailability, and/or reduced sideeffect(s), etc. Optimization can be accomplished through empiricalmodification of the hit structure (e.g., synthesizing compounds withrelated structures and testing them in cell-free or cell-based assays orin non-human animals) and/or using computational approaches. Suchmodification can make use of established principles of medicinalchemistry to predictably alter one or more properties.

In some embodiments, a PLA2G16 inhibitor is modified or incorporates amoiety that enhances cell uptake, stability (e.g., in serum), increaseshalf-life, reduces toxicity or immunogenicity, or otherwise confers adesirable property on the compound. In some embodiments, a PLA2G16inhibitor comprises a protein transduction domain (PTD). A PTD or cellpenetrating peptide (CPP) is a peptide or peptoid that can traverse theplasma membrane of many, if not all, mammalian cells. A PTD can enhanceuptake of a moiety to which it is attached or in which it is present.Often such peptides are rich in arginine. For example, the PTD of theTat protein of human immunodeficiency viruses types 1 and 2 (HIV-1 andHIV-2) has been widely studied and used to transport cargoes intomammalian cells. See, e.g., Fonseca S B, et al., Adv Drug Deliv Rev.,61(11):953-64, 2009; Heitz F, et al., Br J Pharmacol., 157(2):195-206,2009, and references in either of the foregoing, which are incorporatedherein by reference. In some embodiments, a PTD is used to enhance celluptake of a small molecule, siRNA, aptamer, or polypeptide that inhibitsPLA2G16.

In some embodiments, a compound causes a decrease in PLA2G16 level orcatalytic activity of at least 50% when used in a cell-free orcell-based assay at a concentration equal to or less than approximately1 mM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM, or 1 μM. In some embodiments,a compound causes a decrease in PLA2G16 activity of at least 50% (i.e.,a decrease to 50% or less of the activity that would be expected in theabsence of the compound) when used in a cell-free or cell-based assay atlower concentrations, e.g., equal to or less than approximately 500 nM,100 nM, 50 nM, or 10 nM or less. In some embodiments, a compound causesa decrease in PLA2G16 activity of at least 50% when used at aconcentration between 0.1-10 nM. Various methods suitable for assessingPLA2G16 level or activity are mentioned above. In some embodiments, acompound causes a decrease in production or progeny virus of at least50% (i.e., a decrease to 50% or less of the number of progeny virusesthat would be expected in the absence of the compound) or a decrease incytopathic effect of at least 50% when used in a suitable cell culturesystem at a concentration equal to or less than approximately 1 mM, 500μM, 100 μM, 50 μM, 10 μM, 5 μM, or 1 μM. In some embodiments, a compoundcauses a decrease in production or progeny virus or cytopathic effect ofat least 50% when used in a suitable cell culture system at lowerconcentrations, e.g., equal to or less than approximately 500 nM, 100nM, 50 nM, or 10 nM or less. In some embodiments, a compound causes adecrease in production or progeny virus of at least 50% when used in asuitable cell culture system when used at a concentration between 0.1-10nM. Various methods suitable for assessing virus production orcytopathic effect are mentioned above. In other aspects, a compoundcauses a decrease of at least 25%, or at least 75%, or at least 90%, inPLA2G16 level, catalytic activity, and/or production of progeny virus orcytopathic effect.

It is noted that, in general, the PLA2G16 inhibitors and methods of usethereof do not depend on, and are not limited by, the way in which aninhibitor was identified or generated or the components used to identifyor generate the PLA2G16 inhibitor. For example, in certain embodimentsof the invention a PLA216 inhibitor identified using a human PLA2G16polypeptide and/or using human cells is used to treat humans. In certainembodiments of the invention a PLA216 inhibitor identified using a humanPLA2G16 polypeptide and/or using human cells is used to treat non-humananimals, e.g., non-human vertebrate animals. In some embodiments, aPLA216 inhibitor identified using a PLA2G16 polypeptide of a non-humananimal and/or using cells derived from a non-human animal is used totreat non-human animals of that species, different non-human animalspecies, and/or humans. A PLA216 inhibitor that inhibits infection by avirus that infects human cells could be used to treat humans, non-humananimals, or both, in various embodiments of the invention. For example,in certain embodiments a PLA216 inhibitor that inhibits infection by avirus that infects human cells is used to inhibit infection by a virusthat mainly or only infects cells of a non-human animal.

VII. Pharmaceutical Compositions, Methods of Treatment, and OtherApplications

A compound identified, selected, or designed according to a methoddescribed herein can have a variety of uses. In some embodiments, acompound is useful for therapeutic purposes, e.g., as a therapeuticagent for a subject in need of treatment for a viral infection.

In some embodiments, a subject is “suffering from” a viral infectionwhen excessive numbers of a viral population are present in or on theorganism's body and/or when the effects of the presence of a viruspopulation(s) is damaging the cells or other tissue of an organism. Asubject can be “in need of treatment for” a viral infection if, forexample, the subject is suffering from a viral infection or is atincreased risk of developing a viral infection as compared with (i) mostmembers of the general population; and/or (ii) the level of risk thatthe subject typically experiences.

The invention contemplates treatment of a wide variety of viralinfections in human and/or animal subjects, e.g., infection due to anyof the viruses discussed herein. In some embodiments, the virus is apicornavirus, e.g., a cardiovirus, echovirus, enterovirus (e.g., acoxsackievirus, rhinovirus, or echovirus), or hepatovirus, orrhinovirus. In some embodiments, the virus clusters phylogeneticallywithin the enterovirus genus. In some embodiments, the picornavirus isclassified with a species selected from the group consisting of: Humanenterovirus A, Human enterovirus B, Human enterovirus C, Humanenterovirus D, Simian enterovirus A, Bovine enterovirus, Porcineenterovirus B, Human rhinovirus A, Human rhinovirus B and Humanrhinovirus C. In some embodiments, the picornavirus is classified with aspecies selected from the group consisting of: Human enterovirus A,Human enterovirus B, Human enterovirus C, Human enterovirus D, Humanrhinovirus A, Human rhinovirus B and Human rhinovirus C. In someembodiments, the virus is of a serotype that has been deposited at theAmerican Type Culture Collection (ATCC) or National Collection ofPathogenic Viruses (NCPV) of the Health Protection Agency of the UK and,optionally, is available for distribution.

The invention provides methods of treating diseases and medicalconditions resulting from viral infection, e.g., by a picornavirus.Exemplary diseases and conditions include, e.g., asthma exacerbation,bronchiolitis, colitis, common cold, COPD exacerbation, encephalitis,encephalomyelitis, enterocolitis, foot-and-mouth disease,hand-foot-and-mouth disease, gastroenteritis, herpangina, hepatitis,meningitis, meningoencephalitis, myocarditis, pancreatitis, polio, andpneumonia. In some aspects, the invention contemplates ex vivo uses ofthe PLA2G16 inhibitors. For example, organs, tissues, or cells intendedfor use in transplantation (e.g., xenotransplantation or transplantationinto an individual of the same species) can be contacted ex vivo with aPLA2G16 inhibitor, e.g., to reduce the likelihood of transmitting aviral infection to the recipient. In another embodiment, recipients ofan organ, tissue, or cell transplant can be treated with a PLA2G16inhibitor, e.g., to reduce the likelihood of contracting a viralinfection from the transplanted cells, tissues, or organ(s). Suchtreatment could commence prior to, during, or after the transplantprocedure.

In some embodiments, the virus is one for which an effective vaccinedoes not exist, is not in commercial use, or is not widely used. Forexample, coxsackievirus B3 is widespread in the human population andcauses serious diseases such as myocarditis or pancreatitis.Coxsackievirus B4 can cause a broad range of diseases such as asepticmeningitis, meningoencephalitis, myocarditis, hepatitis, pancreatitis,gastroenteritis, necrotizing enterocolitis, and pneumonia. However,despite the clinical significance of these viruses, there is nocommercially available and clinically applicable prophylactic vaccine.Enterovirus 71 is another virus of significant medical importance forwhich a vaccine is not available.

In some embodiments, the virus is one for which an effective vaccine isin commercial use and/or available. Without limitation, the inventivemethods may find use to treat subjects who are unvaccinated or otherwisenon-immune, to treat subjects infected with a strain of virus againstwhich a vaccine may not afford sufficient immunity, etc. In someembodiments, the individual is infected by a vaccine strain, e.g., anattenuated strain. In some embodiments, the invention provides methodsof treating human subjects who may have been exposed to or infected by apoliovirus, e.g., unvaccinated or otherwise non-immune individuals(e.g., immunocompromised individuals), in the setting of a poliooutbreak, individuals who travel to or from a region where polio has notbeen eradicated, etc. In some embodiments, the invention contemplatestreating livestock in need of treatment for foot-and-mouth diseasevirus, e.g., in the setting of a foot-and-mouth disease outbreak.

In some embodiments, a PLA2G16 inhibitor, e.g., a PLA2G16 inhibitoridentified according to the instant invention, can have one or moretherapeutic uses in addition to, or instead of, for treating a viralinfection. In some embodiments, a PLA2G16 inhibitor is useful as atherapeutic agent for a subject in need of treatment for excess bodyfat, a disease associated with excess body fat, or a metabolic disorder.Excess body fat can be a condition of having more body fat than desiredby the subject or having an amount of body fat that is considered withinsound medical judgement to contribute to a disease or to confer anincreased risk of disease. In some embodiments, a compound is useful totreat as atherosclerosis or vascular disease (e.g., cardiovascular orcerebrovascular disease). In some embodiments, the compound is usefulfor treating obesity, e.g., in a subject having a body mass index (BMI)greater than or equal to 30. In some embodiments, a compound is usefulto treat a metabolic disorder, e.g., diabetes (e.g., type II diabetes,also called diabetes mellitus), glucose intolerance, insulin resistance,metabolic syndrome, leptin deficiency, or hypertriglyceridemia.

Inventive methods of treatment can include a step of identifying asubject suffering from or at risk of a viral infection, a step ofidentifying a virus suspected of causing an infection, a step ofselecting a therapeutic agent or combination of agents based at least inpart on the identity or suspected identity of the virus and/or thelocation or characteristics of the infection, and/or a step ofprescribing, providing, or administering a selected agent to thesubject. In certain embodiments of the invention, the method includesdetermining that a subject has a significant likelihood (e.g., at least5%) of suffering from or being at risk of infection by a virus, e.g., apicornavirus. A subject can be “at risk of an infection” in any of avariety of circumstances. “At risk of” implies at increased risk of,relative to the risk such subject would have in the absence of one ormore circumstances, conditions, or attributes of that subject, and/orrelative to the risk that an average, healthy member of the populationwould have and/or relative to the risk that the subject had at aprevious time. The population is typically a group of subjects of thesame species. Examples of conditions that place a subject “at risk”include, but are not limited to, immunodeficiencies (e.g., geneticimmunodeficiencies); prior treatment with antibiotic agent(s) that mayhave reduced or eliminated normal microbial flora; treatment withagent(s) that suppress the immune system (e.g., cancer chemotherapy,immunosuppressive agents); exposure to agents that damage the immunesystem; chronic diseases such as diabetes, COPD, or cystic fibrosis;coexisting or preceding bacterial or fungal infection; surgery or othertrauma; infancy or old age; occupations, events, or living conditionsthat entail exposure to pathogenic viruses, etc., or any other conditionthat within the judgement and skill of the subject's health careprovider place the subject at increased risk. In some embodiments,subject can be at increased risk of developing a viral infection if thesubject has been recently exposed to a pathogenic virus, e.g., thesubject has had contact with an individual known or believed to besuffering from a viral infection (e.g., exposure within the preceding 1,2, 3, or 4 weeks or within the “incubation period” of the virus). In oneembodiment, an incubation period refers to the range of times followingexposure to a virus during which 10%-90% of individuals who developsymptomatic infection would do so.

Any of a variety of methods may be employed to identify a subject inneed of treatment (e.g., a subject at risk of or suffering from a viralinfection) according to the present invention. For example, such methodsinclude clinical diagnosis based at least in part on symptoms, medicalhistory (if available), physical examination, laboratory tests, imagingstudies, immunodiagnostic assays, nucleic acid based diagnostics, and/orisolation and culture of potentially causative viruses from samples,such as blood, urine, sputum, saliva, nasal secretions, stool, synovialfluid, cerebrospinal fluid, bronchealveolar lavage, pus, or any sampleof body fluid, cells, or tissue. In some embodiments, diagnosis can atleast in part be based on serology (e.g., detection of an antibody thatspecifically reacts with the virus). In some embodiments, diagnosis canbe based at least in part on isolating the virus and/or a viral genomeor gene product from the subject. In some embodiment, the sample istested for a viral genome or gene product. For example, PCR or othernucleic acid amplification methods can be used to amplify viral DNA orRNA, which can be detected in a variety of ways such ashybridization-based methods. Multiplexed PCR or other amplificationmethods are useful. Signal amplification assays include branched chainDNA assays and hybrid capture assays. Transcription based amplificationand nucleic acid sequence based amplification (NASBA) may be used.Microarrays, e.g., oligonucleotide micorarrays, can be used. Amicroarray can be a solid phase or suspension array (e.g., amicrosphere-based approach such as the Luminex platform). Immunologicalmethods (e.g., ELISA or particle agglutination) can be used to detectviral antigens, e.g., polypeptides. Labelled compounds that specificallybind to a viral component can be used. In some embodiments, a virus isgrown in cell culture and identified. Identification can be based onmorphology, effect on cultured cells, and/or detection of virus specificnucleic acids and/or polypeptides. In some embodiments, a specific virusis not identified, while in other embodiments a specific virus isidentified.

The compounds and compositions disclosed herein and/or identified orvalidated using a method described herein may be administered by anysuitable means such as orally, intranasally, subcutaneously,intramuscularly, intravenously, intra-arterially, parenterally,intraperitoneally, intrathecally, intratracheally, ocularly,sublingually, vaginally, rectally, dermally, or by inhalation, e.g., asan aerosol. Depending upon the type of condition (e.g., viral infection)to be treated, compounds of the invention may, for example, be inhaled,ingested or administered by systemic routes. Thus, a variety ofadministration modes, or routes, are available. The particular modeselected will depend, of course, upon the particular compound selected,the particular condition being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,may be practiced using any mode of administration that is medically orveterinarily acceptable, meaning any mode that produces acceptablelevels of efficacy without causing clinically unacceptable (e.g.,medically or veterinarily unacceptable) adverse effects. The term“parenteral” includes intravenous, intramuscular, intraperitoneal,subcutaneous, intraosseus, and intrasternal injection, or infusiontechniques. In some embodiments, a route of administration is parenteralor oral. Optionally, a route or location of administration is selectedbased at least in part on the particular viral infection and/or locationof infected tissue. For example, a compound may be delivered to or nearan infected tissue. In some embodiments, inhaled medications are of use.Such administration allows direct delivery to the lung, for example insubjects with a respiratory infection, although it could also be used toachieve systemic delivery. Several types of metered dose inhalers areregularly used for administration by inhalation. These types of devicesinclude metered dose inhalers (MDI), breath-actuated MDI, dry powderinhaler (DPI), spacer/holding chambers in combination with MDI, andnebulizers. In other embodiments, intrathecal administration may be ofuse, e.g., in a subject with a viral infection of the central nervoussystem. Other appropriate routes and devices for administeringtherapeutic agents will be apparent to one of ordinary skill in the art.

Suitable preparations, e.g., substantially pure preparations, of aPLA2G16 inhibitor may be combined with one or more pharmaceuticallyacceptable carriers or excipients, etc., to produce an appropriatepharmaceutical composition. The invention provides a variety ofpharmaceutically acceptable compositions for administration to a subjectcomprising (i) a PLA2G16 inhibitor; and (ii) a pharmaceuticallyacceptable carrier or excipient. The term “pharmaceutically acceptablecarrier or excipient” refers to a carrier (which term encompassescarriers, media, diluents, solvents, vehicles, etc.) or excipient whichdoes not significantly interfere with the biological activity oreffectiveness of the active ingredient(s) of a composition and which isnot excessively toxic to the host at the concentrations at which it isused or administered. Other pharmaceutically acceptable ingredients canbe present in the composition as well. Suitable substances and their usefor the formulation of pharmaceutically active compounds is well-knownin the art (see, for example, “Remington's Pharmaceutical Sciences”, E.W. Martin, 19th Ed., 1995, Mack Publishing Co.: Easton, Pa., and morerecent editions or versions thereof, such as Remington: The Science andPractice of Pharmacy. 21st Edition. Philadelphia, Pa. LippincottWilliams & Wilkins, 2005, for additional discussion of pharmaceuticallyacceptable substances and methods of preparing pharmaceuticalcompositions of various types which are incorporated herein by referencein their entirety).

A pharmaceutical composition is typically formulated to be compatiblewith its intended route of administration. For example, preparations forparenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media, e.g., sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate. fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; preservatives, e.g., antibacterial agents such asbenzyl alcohol or methyl parabens; antioxidants such as ascorbic acid orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. Such parenteral preparations can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.Pharmaceutical compositions and compounds for use in such compositionsmay be manufactured under conditions that meet standards or criteriaprescribed by a regulatory agency. For example, such compositions andcompounds may be manufactured according to Good Manufacturing Practices(GMP) and/or subjected to quality control procedures appropriate forpharmaceutical agents to be administered to humans.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Suitable excipients for oraldosage forms are, e.g., fillers such as sugars, including lactose,sucrose, mannitol, or sorbitol; cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone(PVP). If desired, disintegrating agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereofsuch as sodium alginate. Optionally the oral formulations may also beformulated in saline or buffers for neutralizing internal acidconditions or may be administered without any carriers. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include push fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art.

Formulations for oral delivery may incorporate agents to improvestability in the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, inventive compositions may bedelivered in the form of an aerosol spray from a pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, a fluorocarbon, or a nebulizer. Liquid or dry aerosol(e.g., dry powders, large porous particles, etc.) can be used. Thepresent invention also contemplates delivery of compositions using anasal spray or other forms of nasal administration.

For topical applications, pharmaceutical compositions may be formulatedin a suitable ointment, lotion, gel, or cream containing the activecomponents suspended or dissolved in one or more pharmaceuticallyacceptable carriers suitable for use in such comporisition.

For local delivery to the eye, the pharmaceutically acceptablecompositions may be formulated as solutions or micronized suspensions inisotonic, pH adjusted sterile saline, e.g., for use in eye drops, or inan ointment.

Pharmaceutical compositions may be formulated for transmucosal ortransdermal delivery. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated may be used in theformulation. Such penetrants are generally known in the art. Inventivepharmaceutical compositions may be formulated as suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or as retention enemas for rectal delivery.

In some embodiments, a pharmaceutical composition includes one or moreagents intended to protect the active agent(s) against rapid eliminationfrom the body, such as a controlled release formulation, implants,microencapsulated delivery system, etc. Compounds may be encapsulated orincorporated into particles, e.g., microparticles or nanoparticles.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, PLGA, collagen,polyorthoesters, polyethers, and polylactic acid. Methods forpreparation of such formulations will be apparent to those skilled inthe art. For example, and without limitation, a number of particle-baseddelivery systems are known in the art for delivery of siRNA. Theinvention contemplates use of such compositions. Liposomes or otherlipid-based particles can also be used as pharmaceutically acceptablecarriers.

In some embodiments, the invention provides a pharmaceuticallyacceptable derivative of a PLA2G16 inhibitor, e.g., a PLA2G16 inhibitordescribed herein or identified or validated according to an inventivemethod. According to the present invention, a pharmaceuticallyacceptable derivative of a particular compound includes, but is notlimited to, pharmaceutically acceptable salts, esters, salts of suchesters, or any other adduct or derivative which upon administration to asubject in need thereof is capable of providing the compound, directlyor indirectly. Thus, pharmaceutically acceptable derivatives can includesalts, prodrugs, and/or active metabolites. The term “pharmaceuticallyacceptable salt” refers to those salts which are, within the scope ofsound medical judgment, suitable for use in contact with the tissues ofhumans and/or lower animals without undue toxicity, irritation, allergicresponse and the like, and which are commensurate with a reasonablebenefit/risk ratio. A wide variety of appropriate pharmaceuticallyacceptable salts are well known in the art. Pharmaceutically acceptablesalts include, but are not limited to, those derived from suitableinorganic and organic acids and bases. A pharmaceutically acceptablederivative of a PLA2G16 inhibitor may be formulated and, in general,used for the same purpose(s).

Pharmaceutical compositions of the invention, when administered to asubject, are preferably administered for a time and in an amountsufficient to treat the disease or condition for which they areadministered, e.g., a viral infection. Therapeutic efficacy and toxicityof active agents can be assessed by standard pharmaceutical proceduresin cell cultures or experimental animals. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans or other subjects. Different dosesfor human administration can be further tested in clinical trials inhumans as known in the art. The dose used may be the maximum tolerateddose or a lower dose. A therapeutically effective dose of an activeagent in a pharmaceutical composition may be within a range of about0.001 to about 100 mg/kg body weight, about 0.01 to about 25 mg/kg bodyweight, about 0.1 to about 20 mg/kg body weight, about 1 to about 10mg/kg. Other exemplary doses include, for example, about 1 μg/kg toabout 500 mg/kg, about 100 μg/kg to about 5 mg/kg). In some embodimentsa single dose is administered while in other embodiments multiple dosesare administered. Those of ordinary skill in the art will appreciatethat appropriate doses in any particular circumstance depend upon thepotency of the agent(s) utilized, and may optionally be tailored to theparticular recipient. The specific dose level for a subject may dependupon a variety of factors including the activity of the specificagent(s) employed, severity of the disease or disorder, the age, bodyweight, general health of the subject, etc.

It may be desirable to formulate pharmaceutical compositions,particularly those for oral or parenteral compositions, in unit dosageform for ease of administration and uniformity of dosage. Unit dosageform, as that term is used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active agent(s) calculated toproduce the desired therapeutic effect in association with anappropriate pharmaceutically acceptable carrier. The invention providesa pharmaceutically acceptable unit dosage form containing apredetermined amount of a PLA2G16 inhibitor, such amount beingappropriate to treat a subject in need of treatment for a viralinfection.

It will be understood that a therapeutic regimen may includeadministration of multiple unit dosage forms over a period of time. Insome embodiments, a subject is treated for between 1-7 days. In someembodiments a subject is treated for between 7-14 days. In someembodiments a subject is treated for between 14-28 days. In otherembodiments, a longer course of therapy is administered, e.g., overbetween about 4 and about 10 weeks. In some embodiments a subject istreated at least until at least one symptom or sign of viral infectionhas started to decrease in severity or has significantly decreased inseverity or until a subject is no longer at risk of viral infection. Insome embodiments, treatment may be continued indefinitely, e.g., inorder to achieve prophylaxis. For example, a subject at risk ofrecurrent viral infection or wanting to avoid viral infection may betreated for any period during which such risk exists or the subjectdesires to avoid viral infection. A subject may receive one or moredoses a day, or may receive doses every other day or less frequently,within a treatment period.

In some embodiments, two or more different PLA2G16 inhibitors areadministered. In some embodiments, a PLA2G16 inhibitor is administeredin combination with a second compound useful for treating a viralinfection. The phrase “in combination, as used herein, with regard tocombination treatment means with respect to administration of first andsecond compounds, administration performed such that (i) a dose of thesecond compound is administered before more than 90% of the mostrecently administered dose of the first agent has been metabolized to aninactive form or excreted from the body; or (ii) doses of the first andsecond compound are administered within 48 hours of each other, or (iii)the agents are administered during overlapping time periods (e.g., bycontinuous or intermittent infusion); or (iv) any combination of theforegoing. The compounds may, but need not be, administered together ascomponents of a single composition. In some embodiments, they may beadministered individually at substantially the same time (by which ismeant within less than 10 minutes of one another). In some embodimentsthey may be administered individually within a short time of one another(by which is meant less than 3 hours, sometimes less than 1 hour,apart). The compounds may, but need not, be administered by the sameroute of administration. When administered in combination with a secondcompound, the effective amount of a first compound needed to elicit aparticular biological response may be less or more than the effectiveamount of the first compound when administered in the absence of thesecond compound (or vice versa), thereby allowing an adjustment of theamount dose of the either or both agent(s) relative to the amount thatwould be needed if one compound were administered in the absence of theother. For example, when the compounds of the invention are administeredin combination (e.g., a PLA2G16 inhibitor and a second antiviralcompound), a sub-therapeutic dosage of either of the compounds, or asub-therapeutic dosage of both, may be used in the treatment of asubject in need of treatment for a viral infection. In some embodiments,the two compounds are used in combination, the second antiviral compoundmay in some embodiments be administered at a sub-therapeutic amount toproduce a desirable therapeutic result. A “sub-therapeutic amount” asused herein refers to an amount which is less than that amount whichwould be expected to produce a therapeutic result in the subject ifadministered in the absence of the other compound, e.g., less than arecommended amount. The effects of multiple compounds may, but need notbe, additive or synergistic. One or more of the compounds may beadministered multiple times.

In some embodiments, an antiviral agent known in the art as being usefulfor treating a subject infected with a particular virus, e.g., aPicornavirus, is used as a second compound in combination with a PLA2G16inhibitor. In some embodiments, an antibody that neutralizes or inhibitsthe virus is used. In some embodiments, a compound that inhibits viralfusion is used. In some embodiments a protease inhibitor or kinaseinhibitor is used. In some embodiments an RNAi agent is used, e.g., ansiRNA, e.g., targeting a viral gene. In some embodiments a capsidbinding agent is used. In some embodiments, the second compound is,e.g., ruprintrivir, pleconaril, a pyridazinyl oxime ether, or arbidol.See, e.g., Barnard D L., Current status of anti-picornavirus therapiesCurr Pharm Des.12(11):1379-90, 2006; DePalma, A M, et al., MedicinalResearch Reviews, 28(6): 823-884, 2008.

In some embodiments, a compound that is not sufficiently active to betherapeutically useful is rendered therapeutically useful whenadministered in combination with a PLA2G16 inhibitor. In someembodiments, a lower dose of such compound can be used when administeredin combination with a PLA2G16 inhibitor.

In some embodiments, the invention provides a composition comprising aPLA2G16 inhibitor and a second compound useful for inhibiting a viralinfection, e.g., an infection by a picornavirus. In some embodiments, aunit dosage form comprising the two (or more) agents is provided.

The present invention also provides pharmaceutical packs or kitscomprising one or more containers (e.g., vials, ampoules, bottles)containing a pharmaceutically acceptable PLA2G16 inhibitor and,optionally, one or more other pharmaceutically acceptable ingredients.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceutical products, which notice reflects approval by theagency of manufacture, use or sale for human administration. The noticemay describe, e.g., doses, routes and/or methods of administration,approved indications (e.g., viral infections that the pharmaceuticalcomposition has been approved for use in treating), mechanism of action,or other information of use to a medical practioner and/or patient.Different ingredients may be supplied in solid (e.g. lyophilized) orliquid form. Each ingredient will generally be suitable as aliquoted inits respective container or provided in a concentrated form. Kits mayalso include media for the reconstitution of lyophilized ingredients.The individual containers of the kit are preferably maintained in closeconfinement for commercial sale.

A virus to be inhibited according to the instant invention may infect acell type, organ or organ system of interest. For example, in someembodiments the virus infects cells of the gastrointestinal tract. Insome embodiments the virus infects the liver, e.g., hepatocytes. In someembodiments the virus infects the respiratory system, e.g., cells of theupper and/or lower respiratory tract. In some embodiments the virusinfects muscle cells, e.g., cardiac muscle cells. In some embodimentsthe virus infects the nervous system (e.g., neurons). In someembodiments the virus infects the central nervous system. In someembodiments the virus infects skin cells (e.g., keratinocytes). In someembodiments the virus infects mucosal cells. In some embodiments thevirus infects immune system cells, e.g., lymphocytes or macrophages. Insome embodiments, a virus infection is associated with damage to a celltype, organ, or organ system of interest. Such damage could arise due toinfection of cells by the virus and/or due to immune-mediatedmechanisms.

In some embodiments, a compound is useful for research purposes, e.g.,to further study the role of PLA2G16 in normal physiologic processes orpathologic processes. For example, a compound can be used to furtherstudy the role of PLA2G16 in metabolism and/or in viral infection.

In another aspect, the invention provides a method of generating anon-human multicellular organism, e.g., a non-human animal, e.g., anon-human vertebrate, that has increased resistance to viral infection,e.g., by a picornavirus. In one aspect, the non-human multicellularorganism has reduced endogenous PLA2G16 activity as compared with anormal, non-transgenic organism of the same species. In someembodiments, the organism is a transgenic, non-human vertebrate that hasa targeted insertion into, or deletion of at least part of the PLA2G16gene, so that the animal has reduced expression of functional PLA2G16.In other embodiments, the transgenic non-human animal expresses an RNAiagent, e.g., a shRNA, that reduces PLA2G16 expression. In someembodiments, the organism is not a rodent. In some embodiments theorganism is not a mouse. In some embodiments, the vertebrate is ananimal of commercial importance. For example, the organism maycontribute at least $10,000 to the gross national product of at leastone country and/or be an object of interstate or international commerce.Exemplary animals of commercial importance are, e.g., cows, horses,sheep, goats, pigs, chickens, turkeys, fish. In some embodiments, ananimal is a domesticated animal, e.g., a farm animal, e.g., livestocksuch as a cow, pig, sheep, goat, or horse. In some embodiments, avirus-resistant animal is of a non-domesticated species. Optionally thespecies is endangered. The method can be used to identify individualsthat are resistant to viral infection and have improved likelihood ofsurviving in the wild or in captivity. Animal resistance to viralinfection may reduce the spread of viruses that can infect both animaland human hosts. Mutations or deletions can be engineered using, e.g.,homologous recombination, zinc finger nuclease-mediated recombination,oligonucleotide-mediated gene modification, etc. The transgenic organismcan be generated using standard methods known in the art for generatingsuch organisms. For example, somatic cell nuclear transfer (SCNT) can beused.

In another aspect, the invention provides a method comprisingidentifying a non-human multicellular organism, e.g., a non-humanvertebrate, e.g., a non-human animal, with reduced or absent functionalPLA2G16. In some embodiments, the organism is not a rodent. In someembodiments the animal is not a mouse. In some embodiments, the organismhas reduced expression of PLA2G16. In some embodiments the organismexpresses a functionally inactive variant or fragment of PLA2G16. Forexample, the organism could have a frameshift mutation or a deletion oralteration of at least some residues needed for activity. The organismcan be identified using, e.g., genotyping (e.g., to identify animalsthat have mutations or polymorphisms that result in decreased or alteredPLA2G16) and/or examining expression level in tissues and identifyinganimals with low or absent PLA2G16 expression or activity. In someembodiments, polymorphisms, e.g., single nucleotide polymorphisms (SNPs)that are known in the art are examined. For example, genome projects andother sequencing efforts have identified numerous SNPs in animalgenomes. SNPs, e.g., SNPs located in or near the PLA2G16 gene can beassessed to identify those that are associated with altered, e.g.,reduced or absent, functional PLA2G16. Animals carrying such SNPs can beidentified. In some embodiments, the reduced or absent PLA2G16 occurs inat least some tissues and/or cells that are targets for infection by avirus. In some embodiments, the reduced or absent PLA2G16 occurs in mostor all tissues. Organisms with a desirable trait (e.g., reduced orabsent PLA2G16) can be selected. Standard breeding techniques can beapplied to produce animals with particularly low PLA2G16 expressionand/or activity. For example, standard methods of livestock breedingcould be used. Traditional breeding schemes and/or marker-assistedselection may be employed. In some embodiments, a mutation orpolymorphism is a spontaneously arising mutation, i.e., it is notgenerated by man. In some embodiments, a mutation is generated by man,e.g., using radiation or chemical mutagenesis. Thus the inventionprovides a method of producing a non-genetically modified non-humanorganism, e.g., non-human animal, with reduced or absent functionalPLA2G16. In some embodiments, the method comprising identifying orselecting an organism with reduced or absent functional PLA2G16. In someembodiments, the non-human organism, is produced using selectivebreeding techniques. The invention further provides such organisms andmethods of use thereof.

In some embodiments, a method comprises providing or using an organismwith reduced or absent functional PLA2G16 in agriculture and/or animalhusbandry. The organism can be a genetically modified organism or anon-genetically modified organism. The organism may have reducedlikelihood of infection with a virus and/or may have reduced severity ofinfection. For example, in some embodiments the animal has reducedlikelihood of infection and/or reduced severity of infection by afoot-and-mouth disease virus. In some embodiments the animal has reducedlikelihood of infection and/or reduced severity of infection by a bovineor porcine enterovirus. In some embodiments, the invention provides amethod comprising (a) providing an animal that has reduced or absentfunctional PLA2G16; and (b) engaging in animal husbandry using theanimal. Animal husbandry encompasses the breeding and raising of animalsfor meat or to harvest animal products (such as milk, eggs, or wool) aswell as the breeding and care of species for work and/or companionship.Agriculture refers to the production of food and/or goods throughfarming.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The details of thedescription and the examples herein are representative of certainembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Modifications therein and other uses will occurto those skilled in the art. These modifications are encompassed withinthe spirit of the invention. It will be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention provides all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. It is contemplated that all embodiments described herein areapplicable to all different aspects of the invention where appropriate.It is also contemplated that any of the embodiments or aspects can befreely combined with one or more other such embodiments or aspectswhenever appropriate. Where elements are presented as lists, e.g., inMarkush group or similar format, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. It should be understood that, in general, wherethe invention, or aspects of the invention, is/are referred to ascomprising particular elements, features, etc., certain embodiments ofthe invention or aspects of the invention consist, or consistessentially of, such elements, features, etc. For purposes of simplicitythose embodiments have not in every case been specifically set forth inso many words herein. It should also be understood that any embodimentor aspect of the invention can be explicitly excluded from the claims,regardless of whether the specific exclusion is recited in thespecification. For example, any one or more viral genera, viral species,viruses, assays, compounds, diseases, subjects, or combinations thereof,can be excluded.

Where the claims or description relate to a composition of matter, e.g.,a compound it is to be understood that methods of making or using thecomposition of matter according to any of the methods disclosed herein,and methods of using the composition of matter for any of the purposesdisclosed herein are aspects of the invention, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. Where the claimsor description relate to a method, e.g., a method of identifying acompound, it is to be understood that methods of using the compound, orformulating a composition comprising the compound, as described herein,are aspects of the invention, unless otherwise indicated or unless itwould be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodimentswithin a range of 5% of a number or in some embodiments within a rangeof 10% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue). It should be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It should also beunderstood that unless otherwise indicated or evident from the context,any product or composition described herein may be considered“isolated”.

EXAMPLES Example 1 Characterization and Retroviral Infection of KBM7Subclones

We first characterized a haploid genome setting in human cells that webelieved would be permissive for efficient forward genetic approaches. Asubclone of the CML cell line KBM7 has been described to carry a nearhaploid chromosome set (Kotecki, M., Reddy, P. S., and Cochran, B. H.Isolation and characterization of a near-haploid human cell line. ExpCell Res 252, 273-280, 1999). First we examined if this cell line(generously provided by Dr. B. H. Cochran, Tufts University School ofMedicine, Boston, Mass.) could be easily propagated, was tolerant toviral infection and could be efficiently subcloned. The term “KBM7 cellline” is used herein to refer to this near-haploid cell line or to asubclone thereof. Cells of the KBM7 cell line or a subclone thereof maybe referred to as “KBM7 cells”. KBM7 cells had a high subcloningefficiency (of around ˜80%), and several of the subclones were examinedfurther. The KBM7 subclones proliferated readily with a generation timeof approximately 24 hrs and could be maintained at sparse and very highcell densities (e.g., ˜1×10⁷ cells/ml). Importantly, flow cytometricanalysis indicated that KBM7 subclones had a hypodiploid karyotype ascompared to diploid HCT116 colorectal carcinoma cells. One subclone wasexamined further by 24-color FISH spectral karyotyping and shown to behaploid for all chromosomes except chromosome 8 and to contain aPhiladelphia chromosome (t(9; 22)) characteristic of BCR-ABL transformedchronic myelogenous leukemia cells. See also, PCT Publication No. WO2011/006145 and Carette J E, et al., Haploid genetic screens in humancells identify host factors used by pathogens, Science. 2009 Nov. 27;326(5957):1231-5.

Example 2 Retroviral Infection of KBM7 Cells

We next showed that KBM-7 cells could be infected with retroviruses.Virus was produced by transfection of a GFP expressing retroviral vectorwith packaging vectors in 293T cells (obtained from ATCC). Theretroviral vector was pLIB-GFP (Clontech) but it will be understood thatmany different retroviral vectors could be used. Supernatant containingvirus was used to infect KBM7 cells. To improve the infection efficiencyof KBM7 cells with retroviruses, different conditions were tested.Centrifugation of the cells in a 24-well tissue culture dish for 45minutes at 2,000 pm at room temperature resulted in a 2-fold increase ininfection efficiency compared to no centrifugation. Next the effect ofretronectin, polybrene and protamine sulphate addition was tested,yielding efficiencies of 25%, 33% and 44%, respectively. Eight microgramper milliliter culture medium of protamine sulphate is the preferredaddition. Concentration of virus by ultracentrifugation for 1.5 h at25,000 r.p.m. in a Beckman SW28 rotor dramatically improved infectionrates compared to undiluted virus and was preferred over concentrationby Amicon filters. In conclusion, KBM-7 cells are optimally infectedwhen concentrated virus is used for a spin-infection in the presence ofprotamine sulphate. These subclones could be efficiently (˜70-90%)infected with GFP expressing retroviral or lentiviral viruses that wereVSV-G pseudotyped and maintained high levels of GFP expression forseveral months.

Example 3 Construction of Gene Trap Vectors Containing VectorsContaining Puromycin and GFP Selectable Markers

Retroviral gene trap vectors that contain an inactivated LTR, a strongsplice-acceptor site derived from the long fiber gene of Adenovirusserotype 40 (Carette et al. 2005 The Journal of Gene Medicine 7(8)1053-1062), and either GFP or the puromycin resistance gene (PURO)followed by a SV40 polyadenylation signal were constructed as follows.The coding sequence of the PURO or GFP was obtained by PCR amplificationwith primers containing overhanging ClaI and NheI restriction sites aswell as partial splice acceptor sites:(GFP:5′-GATCGCTAGCCGCATTTCTTTTTTCCAGATGGTGAGCAAGGGCGAGG-3′ and5′-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3′ PURO:5′-GATCGCTAGCCGCATTTCTTTTTTCCAGATGACCGAGTACAAGCCCAC-3′ and5′-GATCGGATCCTCAGGCACCGGGCTTGCGGGTC-3′). These PCR products wereinserted in pEGFPC1 (Clontech) replacing EGFP. Subsequently PCR wasperformed to introduce the complete splice acceptor site and to obtaineither GFP or PURO followed by the poladenylation signal using primerscontaining overhanging ClaI and BamHI sites as well as the 5′ end of thesplice acceptor signal (GFP:5′-GATCATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGATGG-3′ and5′-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3′ PURO:5′-GATCATCGATCGCAGGCGCAATCTTCGCATTTCTTTTTTCCAGATGAC-3′ and5′-GATCGGATCCTTACTTGTACAGCTCGTCCATGC-3′). These PCR products wereinserted in pRETRO-SUPER (Brummelkamp et al. 2002 Cancer Cell.2(3):243-7) replacing the polIII promoter. The resulting plasmids werenamed pGT-GFP and pGT-PURO. Gene trap constructs containing a GFP or apuromycin reporter gene in all three reading frames were generated.

The viral vectors contain an adenoviral splice acceptor site immediatelyupstream of a promoterless reporter and polyadenylation signal so thatvector insertion into an intron of an active gene inactivates the nativelocus, and transcription driven by the gene's promoter results in afusion transcript in which the upstream exon(s) are spliced to the GFPor PURO gene. Since transcription terminates at the inserted polyA site,the resulting fusion transcript encodes a truncated and nonfunctionalversion of the cellular protein and either GFP or PURO, as shownschematically in FIG. 1B for a gene trap vector in which the geneencoding GFP gene serves as a reporter gene.

Example 4 Generation of Mutant Cell Library

To generate a cell library with knock-out alleles in nearly all genes,the near-haploid KBM7-cells were infected with the gene traps generatedas described in Example 3. Gene trap virus was made by transfection of293T cells in T175 dishes with either pGT-GFP or pGT-PURO combined withretroviral packaging plasmids. The virus-containing supernatant wasconcentrated using ultracentrifugation for 1.5 h at 25,000 r.p.m. in aBeckman SW28 rotor. Batches of mutant KBM7 cells are typically made byinfection of one 24-well tissue culture dish containing 1.5 millioncells per well using the method described in Example 2. Cells infectedwith the gene trap containing the puromycin resistance gene wereselected 2 days after infection using 500 ng puromycin per milliliter.After selection by limiting dilution, cells were expanded and frozendown for further screens. The GFP gene trap infected cells were eitherused for screens unselected to negate the gene trap introduced bias foractively expressed genes or were selected using FACS sorting forGFP-expressing cells. In some cases further stratification based on GFPexpression was performed to obtain batches of cells with differentlevels of GFP. To increase the likelihood of identifying genes encodinggene products with a relatively longer half-life, the screens wereperformed on or after day 6 after gene trap infection, thereby allowingthe gene products to dilute during cell proliferation.

Example 5 Generation of a New Cell Type Useful for Haploid Genetics

We generated an additional cell type suitable for haploid genetics usingsomatic cell reprogramming, a method that has recently been describedthat allows reprogramming of the differentiated cell state by, e.g.,introduction of pluripotency-inducing transcription factors such asOCT4, SOX2, KLF4 and c-Myc (Zaehres, H., and Scholer, H. R. (2007).Induction of pluripotency: from mouse to human. Cell 131, 834-835).Introduction of these four transcription factors into KBM-7 cells byretroviral infection (as described in Takahashi, K., et al., Cell,131(5):861-72, 2007) resulted in the formation of adherent cell clones.Some or most of these clones lost the hematopoietic cell surface markersCD43 and CD45. The majority of these cells were not pluripotent. Asubclone was isolated and named “HAP1”. HAP1 cells could be cultured inmedium containing 10% FCS and could be expanded using trypsin. Thesecells were not hematopoietic and the majority of these cells had asingle copy of each chromosome including chromosome 8

In contrast to influenza virus, KBM7 cells cannot be productivelyinfected with poliovirus (FIG. 1B, left panels). HAP1 cells however, arevery susceptible to poliovirus infection and undergo massive cell deathwithin a few days (FIG. 1B, compare upper right and lower right panels).Subsequently, fresh HAP1 cells were infected with our gene trapretroviral construct and exposed to poliovirus. Two resistant colonieswere expanded and the integrations were mapped. Both mutants containedindependent integrations in the known poliovirus entry receptor, PVR,thus explaining their resistance. These results indicated that factorsessential for poliovirus infection can be found through haploid geneticscreens in reprogrammed, non-hematopoietic cell lines derived from KBM7cells, such as HAP1 cells.

Example 6 Identification of PLA2G16 as a Host Factor for Poliovirus

In order to identify new host factors for poliovirus, a larger screenwas undertaken using HAP1 cells (FIG. 1C). Retrovirus was prepared and amutant HAP1 cell library was generated as described in Example 4. Onehundred million mutagenized haploid HAP1 cells were contacted withpoliovirus and resistant colonies were allowed to grow out. To identifygene trap insertion sites, an inverse PCR protocol was adapted for usewith massively parallel sequencing techniques. In order to do so,genomic DNA was isolated from 30 million cells that had been infectedwith a gene trap vector. Four digestion reactions were performed persample, two using NlaIII and two using MseI. Subsequently the digestedDNA was column-purified (Qiagen) and 1 microgram DNA was ligated in avolume of 300 microliter using T4 DNA ligase (NEB) at room temperatureovernight. After another round of column purification the DNA was usedas template for an inverse PCR with outward facing primers. Theoligonucleotides were designed to contain adaptor sequences required foruse with the “Illumina Genome Analyzer”, a massively parallel sequencingplatform. Oligonucleotides used were:5′-AATGATACGGCGACCACCGAGATCTGATGGTTCTCTAGCTTGCC-3′5′-CAAGCAGAAGACGGCATACGACCCAGGTTAAGATCAAGGTC-3′ for templates digestedwith NlaIII. Oligonucleotides used were:5′-AATGATACGGCGACCACCGAGATCTGATGGTTCTCTAGCTTGCC-3′5′-CAAGCAGAAGACGGCATACGACGTTCTGTGTTGTCTCTGTCTG-3′ for templates digestedwith MseI. The four PCR reactions were pooled and used for analysis onan Illumina Genome Analyzer according to manufacturer's protocol andmapped against the human genome. Typically ˜20,000 insertions sitesmapping to different positions on the human genome are obtained fromthis analysis. To facilitate identification of genomic loci that areenriched for gene trap insertions “insertion density” was plotted in agraph. Insertion density was determined for every insertion bycalculating 1/(average distance to three following insertions sites).

The plot in FIG. 2 shows the positions on the human chromosome to whichindividual gene trap mutations were mapped on the x-axis and the inverseof the distance of a particular mutation to its neighbors on the y-axis.Mutations were found to be highly enriched in chromosome 19 in the knownpoliovirus receptor (PVR) and on chromosome 11 in a region that weidentified as the gene encoding the phospholipase PLA2G16. This genecontained 42 independent gene trap insertions.

Example 7 Confirmation that Gene Trap Insertion Ablates PLA2G16Expression

Changing amino acid 113 from C to A (C113A mutation) renders PLA2G16catalytically inactive (Duncan, supra). Retroviral constructs suitablefor expressing wild type or mutant human PLA2G16 in HAP1 cells with orwithout a FLAG tag were generated using standard methods and introducedinto HAP1 cells that contained a gene trap insertion in the PLA2G16locus (PLA2G16^(GT)) The pMX retroviral vector was used expressingFlag-tagged human PLA2G16 and a IRES-Blasticidin selectable marker geneor. For the non-tagged version of PLA2G16 human PLA2G16 cDNA was clonedinto the pBABEpuro retroviral vector). A Western blot was performedusing a polyclonal antibody to PLA2G16 to examine PLA2G16 expression(FIG. 3). PLA2G16 was detected in wild type (WT) HAP1 cells (i.e., HAP1cells that had not been exposed to the gene trap vector) (lane 1). Asexpected, PLA2G16^(GT) cells lacked detectable PLA2G16 (lane 2). As seenin lanes 3-6, PLA2G16 was readily detected in PLA2G16^(GT) cells thathad received a construct encoding PLA2G16 (wild type or C113A mutant).As expected FLAG-tagged PLA2G16 was slightly larger in size thanuntagged PLA2G16 (compare lanes 3 and 4 versus 5 and 6). This experimentdemonstrated that the gene trap had indeed effectively abrogated PLA2G16expression and that the constructs restored PLA2G16 expression whenintroduced into HAP 1 PLA2G16^(GT) cells.

Example 8 Confirmation that Lack of PLA2G16 Renders Cells Resistant toPoliovirus

To confirm that ablating PLA2G16 expression inhibits infection bypoliovirus, haploid PLA2G16^(GT) cells were infected with retrovirusencoding PLA2G16 or a catalytically inactive mutant (containing a C113Aalteration). PLA2G16^(GT) grow robustly in the absence of poliovirus(FIG. 4, left panel). As shown in FIG. 4 (second panel from left),PLA2G16^(GT) cells (containing a PLA2G16 gene trap insertion) also growin the presence of poliovirus. Complementation of PLA2G16 by retroviraloverexpression of wild type PLA2G16 in PLA2G16^(GT) cells restoressensitivity of these cells to poliovirus (second panel from right). Thisrequires the catalytic activity of PLA2G16 because complementation witha catalytic site mutant (C113A) does not restore sensitivity (rightpanel).

FIG. 6A shows the sensitivity of gene trap mutant cells to poliovirus ingraphical form. Cells were infected with indicated MOIs and three daysafter infection viability was measured using an MTT assay. Gene trapinsertion into PLA2G16 renders cells sensitive to infection bypoliovirus, and sensitivity can be restored by expressing wild type butnot catalytically inactive mutant PLA2G16 in the cells. As expected,gene trap insertion into poliovirus receptor renders haploid cellsresistant to poliovirus infection. Gene trap insertion into PLA2G16 hasan essentially equivalent effect to gene trap insertion into poliovirusreceptor.

Example 9 PLA2G16 Insertion Renders Haploid Cells Resistant toCoxsackievirus

FIG. 5 shows effect of coxsackievirus B1 on wild type haploid cells andcells lacking functional PLA2G16. Cells were plated in 24-well wells andmonolayers were treated with coxsackievirus B1 at the indicated MOIs.Four days after infection viable, adherent cells were stained usingcrystal violet. Wild type cells were highly sensitive to the virus atall MOIs tested (top row). Cells mutant for PLA2G16 due to the gene trapinsertion were essentially unaffected by of coxsackievirus B1, even athigh concentrations of virus (second row from top). Complementation ofPLA2G16 by retroviral overexpression restores sensitivity of these cellsto coxsackievirus B1 (third row from top). This requires the catalyticactivity of PLA2G16 because complementation with a catalytic site mutant(C113A) does not restore sensitivity (bottom row). Thus, cellscontaining a PLA2G16 gene trap insertion are resistant to coxsackievirusB1, and sensitivity can be restored by expressing wild type but notcatalytically inactive mutant PLA2G16.

FIG. 6B shows the sensitivity of gene trap mutant cells tocoxsackievirus B1 in graphical form. Cells were contacted with virus atthe indicated MOIs and three days later viability was measured using anMTT assay. Gene trap insertion into PLA2G16 renders cells sensitive toinfection by coxsackievirus B1, and sensitivity can be restored byexpressing wild type but not catalytically inactive mutant PLA2G16 inthe cells. As expected, gene trap insertion into the poliovirus receptordoes not significantly affect sensitivity to coxsackievirus B1.

Example 10 PLA2G16 Knockdown Increases Resistance to Rhinovirus

Ability of RNAi-mediated knockdown of PLA2G16 to inhibit rhinovirusinfection was studied in HeLa cells. PLA2G16 expression was inhibited inHeLa cells using two different siRNAs targeted to PLA2G16, and theability of the cells to survive and proliferate after exposure to humanrhinoviruses HRV-2 and HRV-14 was examined. The siRNAs were Ambion siRNA223200, sequence 5′-CAAGAAACAAGCGACAAAtt-3′ and siRNA 21977 sequence5′-GUACCAGGUCAACAACAAAtt-3′. HeLa cells that had not been transfectedwith siRNA or were transfected with a control siRNA were highlysusceptible to infection by human rhinoviruses HRV-2 and HRV-14 (FIG. 7,two left columns). Knock down of PLA2G16 in Hela cells resulted insignificantly increased resistance to both HRV-2 and HRV-14 (FIG. 7,right two columns). Cells transfected with siRNA targeted to PLA2G16were able to survive and proliferate well following exposure to HRV-2and HRV-14.

We claim:
 1. A method of inhibiting viral infection of a cell comprisingcontacting the cell with a PLA2G16 inhibitor.
 2. The method of claim 1,wherein the virus is a Picornavirus.
 3. The method of claim 2, whereinthe Picornavirus is an enterovirus.
 4. The method of claim 2, whereinthe Picornavirus is a coxsackievirus.
 5. The method of claim 2, whereinthe Picornavirus is a hepatovirus.
 6. The method of claim 2, wherein thePicornavirus is a rhinovirus.
 7. The method of claim 1, wherein the cellis a vertebrate cell.
 8. The method of claim 1, wherein the cell is amammalian cell.
 9. The method of claim 1, wherein the cell is a humancell.
 10. The method of claim 1, wherein the inhibitor inhibitsexpression of PLA2G16.
 11. The method of claim 1, wherein the inhibitorinhibits enzymatic activity of PLA2G16.
 12. A method of treating a viralinfection in a subject, the method comprising administering a PLA2G16inhibitor to a subject in need of treatment for a viral infection. 13.The method of claim 12, wherein the viral infection is a Picornavirusinfection.
 14. The method of claim 13, wherein the Picornavirus is anenterovirus.
 15. The method of claim 13, wherein the Picornavirus is acoxsackievirus.
 16. The method of claim 13, wherein the Picornavirus isa hepatovirus.
 17. The method of claim 13, wherein the Picornavirus is arhinovirus.
 18. The method of claim 12, wherein the subject is avertebrate.
 19. The method of claim 12, wherein the subject is a mammal.20. The method of claim 12, wherein the subject is human.
 21. The methodof claim 12, wherein the inhibitor inhibits expression of PLA2G16. 22.The method of claim 12, wherein the inhibitor inhibits enzymaticactivity of PLA2G16.
 23. A method of identifying a candidate antiviralcompound comprising steps of: (a) providing a composition comprising aPLA2G16 polypeptide and a test compound; (b) determining whether thetest compound inhibits the PLA2G16 polypeptide, wherein if the compoundinhibits the PLA2G16 polypeptide, the compound is identified as acandidate antiviral compound.
 24. The method of claim 23, wherein step(b) comprises determining whether the test compound inhibits expressionof the PLA2G16 polypeptide.
 25. The method of claim 23, wherein step (b)comprises determining whether the test compound inhibits an enzymaticactivity of the PLA2G16 polypeptide.
 26. The method of claim 25, whereinthe enzymatic activity is phospholipase A2 activity.
 27. The method ofclaim 23, wherein the composition of step (a) is a cell-free compositioncomprising purified PLA2G16; and step (b) comprises determining whetherthe test compound inhibits enzymatic activity of PLA2G16.
 28. The methodof claim 23, wherein the composition of step (a) comprises a cell thatexpresses a PLA2G16 polypeptide, and wherein step (b) comprisesdetermining whether the test compound inhibits expression or enzymaticactivity of PLA2G16.
 29. The method of claim 23, wherein if thecompounds inhibits the PLA2G16 polypeptide, the compound is identifiedas a candidate antiviral compound useful for inhibiting viral infectionby a Picornavirus.
 30. The method of claim 23, further comprisingassessing the ability of the compound to inhibit viral infection of acell or subject.
 31. The method of claim 23, further comprising the stepof contacting a cell with the compound and a virus, wherein the cellwould be susceptible to the virus in the absence of the compound. 32.The method of claim 23, further comprising the step of administering thecompound to a subject, wherein the subject would be susceptible toinfection by the virus in the absence of the compound.
 33. The method ofclaim 23, further comprising the step of contacting a cell that isinfected by the virus with the compound.
 34. The method of claim 23,further comprising the step of administering the compound to a subject,wherein the subject is infected by a virus.
 35. A method of validating acandidate antiviral compound comprising steps of: (a) providing acandidate antiviral compound identified according to the method of claim23; and (b) determining whether the compound inhibits infection of acell or organism by a virus, wherein if the compound inhibits infectionof a cell or organism by the virus, the compound is validated as anantiviral compound.
 36. The method of claim 35, wherein the virus is aPicornavirus.
 37. A composition comprising: (a) a PLA2G16 inhibitor; (b)a virus; and (c) a population of cells.
 38. The composition of claim 37,wherein the virus is present at a multiplicity of infection (MOI) of atleast 0.01.
 39. The composition of claim 37, wherein the virus is aPicornavirus.
 40. The composition of claim 37, wherein the cells are inculture.
 41. The composition of claim 37, wherein the cells arevertebrate cells.
 42. The composition of claim 37, wherein the cells aremammalian cells.
 43. The composition of claim 37, wherein the cells arehuman cells.
 44. The composition of claim 37, wherein at least some ofthe cells are infected by the virus.
 45. The composition of claim 37,wherein the PLA2G16 inhibitor binds to PLA2G16.
 46. The composition ofclaim 37, wherein the PLA2G16 inhibitor inhibits expression of PLA2G16.47. The composition of claim 37, wherein the PLA2G16 inhibitor inhibitsan enzymatic activity of PLA2G16.
 48. The composition of claim 37,wherein the PLA2G16 inhibitor is a small molecule.
 49. The compositionof claim 37, wherein the PLA2G16 inhibitor is present in an amountsufficient to detectably inhibit infection of the cells by the virus.50. A composition comprising a PLA2G16 inhibitor, wherein thecomposition is useful for treating a viral infection in a subject. 51.The composition of claim 50, wherein the PLA2G16 inhibitor binds toPLA2G16.
 52. The composition of claim 50, wherein the PLA2G16 inhibitorinhibits expression of PLA2G16.
 53. The composition of claim 50, whereinthe PLA2G16 inhibitor inhibits an enzymatic activity of PLA2G16.
 54. Thecomposition of claim 50, wherein the PLA2G16 inhibitor is a smallmolecule.
 55. The composition of claim 50, wherein the viral infectionis a picornavirus infection.
 56. The composition of claim 50, whereinthe subject is a vertebrate.
 57. The composition of claim 50, whereinthe subject is a mammal.
 58. The composition of claim 50, wherein thesubject is human.
 59. A near-haploid mammalian cell that has a mutationin a gene that encodes PLA2G16.
 60. The near-haploid mammalian cell ofclaim 59, wherein the cell expresses a mutant form of PLA2G16.
 61. Thenear-haploid mammalian cell of claim 60, wherein the cell expresses amutant form of PLA2G16, wherein the mutant form has reduced catalyticactivity as compared with the non-mutant form.
 62. A method ofidentifying a non-human multicellular organism with increased resistanceto infection by a virus, the method comprising determining whether theorganism has reduced PLA2G16 expression or activity, wherein if theorganism has reduced PLA2G16 expression or activity, the organism hasincreased resistance to infection by a virus.
 63. The method of claim62, wherein the virus is a Picornavirus.
 64. The method of claim 62,wherein the organism is a commercially important vertebrate animal. 65.A method comprising: (a) providing a multicellular organism with reducedor absent functional PLA2G16; and (b) using the organism in agricultureand/or animal husbandry.
 66. The method of claim 65, wherein theorganism is a commercially important vertebrate animal.
 67. The methodof claim 65, wherein the organism is not genetically modified.
 68. Themethod of claim 65, wherein the organism has increased resistance toinfection by a Picornavirus relative to an organism that does not havereduced or absent functional PLA2G16.
 69. A farm animal having reducedor absent functional PLA2G16, wherein the animal has increasedresistance to infection by a virus.
 70. The farm animal of claim 69,wherein the virus is a Picornavirus.
 71. The farm animal of claim 69,wherein the farm animal is a cow, pig, sheep, goat, horse, chicken, orturkey.